Targeting Pim Kinases and DAPK3 to Control Hypertension

Article

Targeting Pim Kinases and DAPK3 to Control

Hypertension

Graphical Abstract

Authors

David A. Carlson, Miriam R. Singer,

Cindy Sutherland, ...,

Matthew A. Sparks,

Justin A. MacDonald,

Timothy A.J. Haystead

Correspondence

timothy.haystead@duke.edu

In Brief

Carlson et al. use crystal structure-guided

medicinal chemistry techniques to

develop a dual Pim/DAPK3 inhibitor

(HS56) that reduces myosin

phosphorylation and contractility in

smooth muscle. Their ﬁndings reveal the

contribution of Pim kinases to the

pathology of hypertension, suggesting a

novel multi-target engagement strategy

for molecularly targeted antihypertensive

medications.

Highlights

d

A dual Pim/DAPK3 inhibitor (HS56) was developed

Pim kinases directly phosphorylate smooth muscle targets

Pim/DAPK3 inhibition reduces phospho-LC20 and

contractility in smooth muscle

d

HS56 decreased blood pressure in spontaneously

hypertensive RenTG mice

Carlson et al., 2018, Cell Chemical Biology 25, 1–13

September 20, 2018 ª 2018 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2018.06.006

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Cell Chemical Biology

Article

Targeting Pim Kinases and DAPK3

to Control Hypertension

David A. Carlson,¹Miriam R. Singer,¹Cindy Sutherland,²Clara Redondo,³Leila T. Alexander,³Philip F. Hughes,¹

Stefan Knapp,^3,4Susan B. Gurley,⁵Matthew A. Sparks,⁵Justin A. MacDonald,²and Timothy A.J. Haystead^1,6,

*

¹Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA

²Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary,

AB T2N 4Z6, Canada

³Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK

⁴Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany

⁵Division of Nephrology, Department of Medicine, Duke University and Durham VA Medical Centers, Durham, NC 27710, USA

⁶Lead Contact

*Correspondence: timothy.haystead@duke.edu

https://doi.org/10.1016/j.chembiol.2018.06.006

SUMMARY

smooth muscle (VSM) hypercontractility (Bai et al., 2013; Uehata

et al., 1997). VSM contractility can be modulated by antihyper-

Sustained vascular smooth muscle hypercontractil- tensive medications that attempt to restore normal BP and

reverse remodeling (Briet and Schiffrin, 2013); for example, NO

donors, vasodilating b-blockers, Ca²⁺channel blockers, and

renin-angiotensin system (RAS) blockers (Briet and Schiffrin,

ity promotes hypertension and cardiovascular

disease. The etiology of hypercontractility is not

completely understood. New therapeutic targets

remain vitally important for drug discovery. Here we

report that Pim kinases, in combination with

DAPK3, regulate contractility and control hyperten-

sion. Using a co-crystal structure of lead molecule

(HS38) in complex with DAPK3, a dual Pim/DAPK3 in-

2013; Garcia-Donaire et al., 2011). However, despite wide-

spread usage, antihypertensive medications restore normal BP

in fewer than 50% of patients (Crowley and Coffman, 2014; Gu

et al., 2012). Side effects (Wiysonge and Opie, 2013) and drug

resistance (Calhoun et al., 2008) further reduce the beneﬁts of

current drugs.

hibitor (HS56) and selective DAPK3 inhibitors (HS94

and HS148) were developed to provide mechanistic

Approximately 1% of chronic hypertension patients experi-

ence acute, rapid elevation of BP (>160/100 mmHg) with poten-

insight into the polypharmacology of hypertension. tially fatal end-organ damage, termed hypertensive emergency

(HE) (Pollack and Rees, 2008; Tulman et al., 2012). Clinical man-

agement of HE is difﬁcult because immediate therapeutic

In vitro and ex vivo studies indicated that Pim kinases

directly phosphorylate smooth muscle targets and

that Pim/DAPK3 inhibition, unlike selective DAPK3

inhibition, signiﬁcantly reduces contractility. In vivo,

HS56 decreased blood pressure in spontaneously

hypertensive mice in a dose-dependent manner

without affecting heart rate. These ﬁndings suggest

including Pim kinase inhibition within a multi-target

lowering of BP can cause end-organ ischemia. For efﬁcient titra-

tion, the ideal HE therapeutic would be dose dependent, with

rapid onset and rapid clearance. However, most HE therapies,

such as nitroprusside and dihydropyridine analogs, are limited

by poor pharmacokinetics or narrow therapeutic windows and

are not applicable across the broad range of HE comorbidities.

Clevidipine, a Food and Drug Administration (FDA)-approved

Ca²⁺channel antagonist for the treatment of HE, has high thera-

peutic value; however, side effects (e.g., atrial ﬁbrillation and

engagement strategy for hypertension manage-

ment. HS56 represents a signiﬁcant step in the devel-

opment of molecularly targeted antihypertensive sinus tachycardia [Keating, 2014], resistance, and drug interac-

tions) have recently been observed (Jacklen et al., 2014). New

therapeutic targets and medications for modulating VSM

contractility to control both acute and chronic hypertension

medications.

INTRODUCTION

remain an urgent necessity (Pijacka et al., 2016; Retailleau

et al., 2015).

Hypertension, or elevated arterial blood pressure (BP), affects

Most antihypertensive medications act at the receptor or

one-third of American adults, whose risk of acute and chronic second messenger level (RAS, adrenoceptors, Ca²⁺, Na⁺/K⁺)

morbidity correlates directly with increasing BP (Lifton et al., and therefore have broad physiological effects. A more focused

2001). Chronic hypertension-related pathologies—cardiac and and largely unexplored strategy involves inhibition of down-

pulmonary arterial hypertrophy (PAH) (Voelkel and Tuder, 1997; stream regulators of VSM contraction. In VSM, contractility

Zanchetti, 2010), kidney disease (Crowley and Coffman, 2014), is governed by homeostatic control of myosin light chain

heart disease, and stroke (Lawes et al., 2008)—are invariably (LC20) phosphorylation (MacDonald et al., 2016; Somlyo and

associated with hypertrophic remodeling (Briet and Schiffrin, Somlyo, 1994). LC20 phosphorylation by myosin light-chain ki-

2013; Sonoyama et al., 2007) resulting from sustained vascular nase (MLCK) induces contraction and is reversed by myosin

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Figure 1. Structure and In Vitro Characterization of Lead Compound and Second-Generation Inhibitors

(A) Lead compound HS38 with zones of variability (1); dual Pim/DAPK3 inhibitor HS56 (2); DAPK3 inhibitors HS94 (3) and HS148 (4); and inactive

analog HS182 (5).

(legend continued on next page)

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light-chain phosphatase (MLCP) (Butler et al., 2013; Hartshorne (Figure 1A). As a speciﬁc inhibitor of DAPKs and Pim-3, HS38

et al., 1998; Sherry and Hartshorne, 1980). Whereas MLCK displayed higher speciﬁcity and greater potency, and lacks

is dependent upon intracellular free Ca²⁺([Ca²⁺]_i), MLCP is chemical liabilities inherent in other DAPK inhibitors (Carlson

[Ca²⁺

] independent and controlled by its myosin targeting et al., 2013). In VSM tissues, HS38 delayed agonist-induced

i

subunit (MYPT1). MYPT1 phosphorylation inactivates MLCP, contraction and suppressed Ca²⁺sensitization (MacDonald

suppresses myosin dephosphorylation, and triggers hypercon- et al., 2016).

tractility in the absence of elevated [Ca²⁺]_i(Ca²⁺sensitization)

Herein we report an investigation into Pim kinases as modula-

(Goulopoulou and Webb, 2014; Kitazawa et al., 1989). Sustained tors of VSM contractility. Using a co-crystal structure of HS38 in

Ca²⁺sensitization induces remodeling and disease progression complex with DAPK3, a second-generation dual Pim/DAPK3 in-

(Sonoyama et al., 2007); therefore, MYPT1 and its immediate hibitor HS56 (2) and selective DAPK3 inhibitors HS94 (3) and

regulators represent critical points of intervention for antihyper- HS148 (4) were developed and used to gain mechanistic insight

tensive medications.

into the contribution of Pims to VSM contraction and BP modu-

Rho-associated kinase (ROCK)-induced MYPT1 phosphoryla- lation. Characterization in vitro, ex vivo, and in vivo suggests that

tion and Ca²⁺sensitization has been well characterized (Bai Pims directly modulate VSM contractility and, together with

et al., 2013; Uehata et al., 1997; Walsh and MacDonald, 2016). DAPK3, represent polypharmacological targets for the treatment

Although ROCK inhibition normalizes BP in hypertensive rodent of chronic hypertension.

models, the suboptimal selectivity of many ROCK inhibitors

diminishes their therapeutic value (Bain et al., 2007). We RESULTS

have instead focused on death-associated protein kinase 3

(DAPK3), also named zipper-interacting protein kinase (ZIPK) Discovery of HS56, HS94, and HS148

(Carlson et al., 2013; Haystead, 2005; MacDonald et al., Small-molecule inhibitors are essential for understanding the un-

2001a, 2016; Turner et al., 2015), a Ser/Thr kinase and down- derlying mechanisms of Pim and DAPK activity within VSM and

stream target of ROCK that promotes Ca²⁺sensitization in vivo for determining their therapeutic value in hypertensive models.

(Borman et al., 2002) by phosphorylating both MYPT1 and the HS56, HS94, and HS148 were developed by the introduction

MYPT1-inhibitor CPI-17 (MacDonald et al., 2001b). The DAPK of diverse functionality at three variable zones around the pyra-

family (DAPKs 1, 2, and 3) possess identical nucleoside binding zolo[3,4-d]pyrimidinone scaffold of HS38 (Figure S2). Resulting

residues and are not readily discriminated by ATP competitive analogs (Table S1) were evaluated using a radioactive [³²P]ATP

inhibitors. However, compared with other kinase families, the ﬁlter-binding kinase inhibition assay (Hastie et al., 2006) to deter-

DAPK3 catalytic domain contains numerous structural features mine inhibitory activity ([Analog] = 10 mM) versus DAPK3, Pim-1,

that render it amenable to selective inhibition (Temmerman Pim-2, and Pim-3 (Figure 1B, a subset of Figure S3). A subset of

et al., 2013). Within the DAPK family only DAPK3 is expressed analogs displaying >80% inhibitory activity toward DAPK3 were

in VSM, where its effects are regulated by phosphorylation at titrated in the same assay (Figure 1C, a subset of Figures S4A–

Thr180, Thr225, and Thr265 (Graves et al., 2005). S4D) to determine inhibition constants (K_i) (Figure 2A, a subset

Within the kinome, the catalytic domain of DAPK3 is highly of Figure S4F).

similar in sequence and structure to Pim kinases (Manning

Key improvements in DAPK3 potency resulted from HS94 (3)

et al., 2002). These Ser/Thr kinases have been previously asso- and HS148 (4): K_i= 126 nM and 119 nM, respectively. Dual

ciated with cell survival and proliferation by regulation of Pim/DAPK3 inhibitor HS56 (2) maintained potency toward

apoptosis and division (Bachmann and Moroy, 2005; Mukaida DAPK3 (K_i= 315 nM) and was most potent versus Pim-3 (K_i=

et al., 2011; Nawijn et al., 2011). As such, they have been the 72 nM) with micromolar potency toward Pim-1 (K_i= 1.5 mM)

focus of discovery efforts for cancer therapeutics (Braso-Mari- and Pim-2 (K_i= 17 mM). These second-generation inhibitors

stany et al., 2016; Pogacic et al., 2007). In addition to cancer tis- have high therapeutic potential and served as key molecular

sues, the Pims (Pim-1, -2, and -3) are constitutively active and probes to investigate the effects of Pim/DAPK3 inhibition and se-

transcriptionally regulated (Willert et al., 2010) within human car- lective DAPK3 inhibition on VSM contractility and hypertension.

diac, skeletal, and VSM tissues (Muraski et al., 2007; Renard

HS56 displayed a high degree of selectivity for DAPKs and

et al., 2013) (Figure S1). Pim-1 plays a signiﬁcant role in VSM re- Pims. HS56 was evaluated in an active site-directed competition

modeling (Liang and Li, 2014) and in the pathogenesis of PAH binding assay (KINOMEscan; DiscoverX, Fremont, CA). Of the

(Paulin et al., 2011a, 2011b); however, these effects were not 468 kinases assayed, HS56 competitively inhibited only seven

previously linked to contractility, but rather to pro-proliferative with %Control < 10 (Figure 2B). This subset contained ﬁve

effects of the kinase (Katakami et al., 2004; Zippo et al., 2004).

desired targets (Pim-1, Pim-3, DAPK-1, -2, and -3) and two

We reasoned that if Pims modulate VSM contractility, then off-target interactions; non-receptor tyrosine-protein kinase 2

multi-target Pim/DAPK3 inhibition may substantially reduce BP (TYK2) and cyclin-G-associated kinase (GAK) (Figure 2C).

in vivo. To test this hypothesis, we developed key inhibitors for TYK2 is a member of the JAK family and is not likely relevant

probing Pim/DAPK3 inhibition from our lead molecule, HS38 (1) to smooth muscle contraction (Hubbard, 2018). Moreover,

(B) Heatmap representing residual kinase activity following treatment with all analogs (10 mM, n = 2) in a radioactive [³²P]ATP ﬁlter-binding kinase assay. (À), no

inhibitor. (+), no kinase.

(C) Kinase inhibition isotherms generated from titrating compounds 1–4 against DAPK3, Pim-1, Pim-2, and Pim-3 using radioactive [³²P]ATP ﬁlter-binding assay.

Data points represent mean SEM (n = 2).

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Figure 2. Potency and Selectivity

(A) Inhibition constants (K_i) for analogs 1–5. EC₅₀

values derived from kinase inhibition isotherms

(Figure 1C) were converted to K_ivalues using the

Cheng-Prusoff equation (Cheng and Prusoff, 1973)

(Figure S4G).

(B–D) Primary KINOMEscan proﬁling of HS56 us-

ing a competition binding assay. (B) Full kinome

proﬁle of HS56. %Control = 100 3 (HS56 signal À

positive control)/(negative control

À

positive

control). (C) Subset of data from (B) showing VSM

active kinase families (green) and kinases for which

%Control is <10 (red). (D) Dendrogram of human

kinases showing a subset of data from (B).

(E) Inhibition of ionotropic and G-protein-coupled

receptors by HS56. Data points represent mean

SEM (n = 4).

afﬁnity between HS38 and DAPK3 and

delineated uniquely targetable features.

Our structure is consistent with the pub-

lished DAPK3 catalytic domain (PDB:

1P4F) (Velentza et al., 2003) with a root-

˚

mean-square deviation (RMSD) of 1.15 A

considering 252 Ca atoms. DAPK3

adopts a bilobal structure typical of ki-

nases with a smaller b-stranded N-termi-

nal lobe and a larger a-helical C-terminal

lobe. HS38 sits within the ATP binding

pocket at the junction of these two lobes.

DAPK3 contains uncommon features

that render it distinct from other members

of the human kinome (Figure S5A). Regu-

latory phosphorylation sites within its

ATP-binding pocket are absent. Its gate-

keeper residue (Leu93) is conserved in

HS56 displayed afﬁnity toward inactive TYK2 (JH2domain- only 18% of kinases (Moffat et al., 2011; Yokoyama et al.,

pseudokinase) and not catalytically active TYK2 (JH1domain- 2015). The adenine binding pocket hinge region contains a

catalytic) (%Control = 92) (Table S3). GAK regulates endocytosis non-conserved residue (Glu94), which H-bonds to adenine-

and uncoating of clathrin-coated vesicles (Neveu et al., 2015) NH₂of ATP, forming a canonical donor-acceptor pair. Addition-

and is also not likely to regulate VSM contraction. Additionally, ally, the phosphate binding pocket contains a key residue

TKY2 and GAK are located on remote branches of the human ki- (Lys42), which forms only one H bond to ATP instead of two (Ter-

nome dendrogram and are dissimilar in sequence to members of eshko et al., 2001). Thus, ATP competitive inhibitors may target

the CAMK subgroup (Figure 2D). The quantitative selectivity DAPK3 separately from other closely related kinases (Huang

score, S(10) (number of non-mutant kinases with %Control <10 )/ et al., 2010), including the Pims, despite structural similarities

(number of non-mutant kinases tested) for HS56 is similar to la- and proximity on adjacent sub-branches of the CAMK region

patinib (Karaman et al., 2008), the most selective, FDA approved of the kinome dendrogram (Manning et al., 2002).

kinase inhibitor (S(10)_HS56= 0.017 and S(10)_lapatinib= 0.010).

Analysis of non-covalent interactions between HS38 and

To rule out activity on cell surface receptors, we assayed HS56 DAPK3 (Figure 3B) suggested water-mediated H bonds to (1)

by the PDSP database (Department of Pharmacology, UNC, hinge region backbone between aryl chlorine and Val96/Gly99;

Chapel Hill, NC) against ionotropic and G-protein-coupled (2) adenine binding pocket backbone between pyrazole N2

receptors (Figure 2E). HS56 displayed no signiﬁcant (>50%) inhi- and Glu94/Val96; (3) phosphate binding pocket between pyrimi-

bition or activation of nicotinic, adrenergic, or muscarinic recep- dinone oxygen and Glu64(N )³/Phe162; and (4) between thioether

tors at 10 mM.

carboxamide and Asn144/Asp161. A single direct H bond was

present between the thioether and Lys42 (Figure 3C). Notably

absent are canonical donor-acceptor H bonds to the hinge re-

Structural Basis of DAPK3 and Pim Inhibition

The crystal structure of human DAPK3 catalytic domain in com- gion backbone (Figure S5B). The HS38 chlorophenyl ring is

plex with HS38 (PDB: 5VJA) (Figure 3) (CRELUX, Martinsried, sandwiched between Leu19 and Ile160, providing hydrophobic

Germany) elucidated intermolecular forces driving nanomolar stabilization.

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Figure 3. Crystal Structure of DAPK3 in Complex with HS38 Deﬁnes Domains of Interaction

(A) Overlaid ribbon and surface diagrams of the DAPK3 catalytic domain (red). HS38 (orange) is bound to the ATP binding cleft (cyan).

(B) Detailed view of HS38 (orange) with interacting resides (cyan sticks), H bonds (black lines), and water molecules (red spheres).

(C) 2D depiction with color-coded domains of DAPK3-HS38 interaction: direct H bonding (dashed black line), hydrophobic interactions (magenta). For data

collection and reﬁnement, see Table S2.

Pim kinases share a high degree of sequence similarity within

HS94 and HS148 are more potent toward DAPK3 and less

the Pim family (Bachmann and Moroy, 2005), but only ꢀ30% potent toward all Pims. Both have additional hydrophobic sur-

overall sequence similarity with other kinases (Jacobs et al., face area (a-dimethyl and a-ethyl) on zone 2 that likely stabilizes

2005). The activation loop of Pim-1 contains phosphorylation the more conﬁned hydrophobic loop of DAPK3. Both analogs

sites; however, Pim kinases are constitutively active, regardless lack an aryl chloride substituent (zone 1), suggesting that smaller

of activation loop phosphorylation, due to stabilizing interactions meta-aryl substitution may increase potency toward DAPK3

with the catalytic loop (Merkel et al., 2012; Qian et al., 2005).

while larger substituents may orient favorably within the hinge re-

Our DAPK3 structure was compared with Pim-1 to give struc- gion bulge of the Pims. The loss of water mediated H-bonding

tural insight into the potency of key analogs. The Pim-1 ATP- between HS94/HS148 and Gly99/Val96 of DAPK3 is likely offset

binding cleft shares ꢀ75% sequence similarity with DAPK3 (Fig- by additional hydrophobic interactions.

ures 4A and 4B). Although DAPK3 overlays closely with Pim-1

Zone 3 contains an oxygen substituent in the 4-position that

˚

(RMSD between 171 atom pairs is 1.14 A), detailed structural participates in two water-mediated H bonds to DAPK3, contrib-

analysis revealed several targetable features (Figures 4C and uting substantially to the energetics of binding. This pyrimidone

4D). Like DAPK3, Pim-1 adopts a DFG-in conformation. The oxygen is otherwise surrounded by hydrophobic residues.

glycine-rich hydrophobic pocket at the entrance region of Replacement with -NH₂or alkylated amines proved deleterious

Pim-1 (residues 44–52) is signiﬁcantly wider than in DAPK3 (res- to inhibitor potency and led to inactive analog HS182, used

idues 19–27) (Jacobs et al., 2005; Yokoyama et al., 2015) giving here as a negative control.

Pim-1 greater accessibility. The narrower loop of DAPK3 may

In general, numerous hydrophobic residues on DAPK3 are re-

provide hydrophobic stabilization with the propanamide methyl placed with hydrophilic, basic, acidic, and proline residues on

of HS38, a potential structural basis for selectivity. The wider Pim-1. Altogether, these combined structural differences likely

loop on Pim-1 likely accommodates a wider range of thioether account for the higher potency of HS94 and HS148 for DAPK3

side chains. An unusual hinge region bulge, conserved within and the loss of potency toward Pims. HS148 and HS94 were

Pim kinases, forms only one backbone H bond with ATP due essential for determining the effect of DAPK3 inhibition on VSM

to unique proline resides (Pro123 and Pro125). This unique hinge contractility in the absence of Pim inhibition. HS56, our most

likely accommodates structural diversity different from that of potent Pim inhibitor, remains potent toward DAPK3 and was a

DAPK3. Constrained rotation also creates an unusual hinge key molecular probe for elucidating the effect of dual Pim/

bulge potentially weakening hydrophobic stabilization with the DAPK3 inhibition on VSM contractility and hypertension.

chlorophenyl moiety of HS38, which packs tightly into the hydro-

phobic cleft of DAPK3.

Modulation of VSM Contractility

Precise balancing of LC20 phosphorylation modulates the

kinetics and intensity of smooth muscle contraction (Walsh,

Structural Insight into Analog Potency

All analogs share an identical pyrazolo[3,4-d]pyrimidin-4-one 2011). LC20 phosphorylation and contraction dynamics were

core scaffold with three zones of variability (Figure 1A). HS56 characterized in excised arterial VSM. Rat caudal arterial smooth

displays enhanced potency toward Pims and is identical to muscle strips were cleaned of adventitia and endothelium

HS38 in all respects except its cyanomethyl thioether substituent denuded. Contraction was induced with the phosphatase

(zone 2), which likely orients readily within the wider glycine-rich inhibitor, calyculin A (Cla) (Figure 5A). Pretreatment with selective

loop of Pims.

DAPK3 inhibitors (HS94, HS148), Pim-3/DAPK3 inhibitor (HS38),

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Figure 4. Structural Analysis of DAPK3 and Pim-1 Reveals Uniquely Targetable Features

(A) CLUSTAL 2.1 multiple sequence alignment of DAPK3, Pim-1, Pim-2, and Pim-3.

(B) Table of amino acid substitutions within the ATP binding pockets of DAPK3, Pim-1, Pim-2, and Pim-3.

(C) Overlaid catalytic domains of DAPK3-HS38 (red) and Pim-1 bound to AMPPNP (gray, ligand not shown, PDB: 1XR1) (Qian et al., 2005). HS38 (orange) sits

within the ATP binding cleft of DAPK3 (cyan). Unique residues within the Pim-1 ATP binding pocket (yellow).

(D) Overlaid ATP binding pockets of DAPK3 (cyan) and Pim-1 with conserved residues (gray) and non-conserved residues (yellow, labels). HS38 (orange) is bound

to DAPK3 and water molecules (red spheres) by H bonds (black lines).

and inactive control (HS182) resulted in similar contractile mor- ure 5D). Similar trends were observed for t_{1/2 max}after contrac-

phologies; Cla-induced force increased to a stable plateau tion and latency (Figures S6A and S6B). All three analogs were

greater than K⁺-induced reference force. Dramatic differences active at concentrations as low as 1 mM, ex vivo potency typical

were observed after pretreatment with HS56; force increased of bioavailable, ATP-competitive kinase inhibitors with nanomo-

to an unstable maximum never greater than K⁺reference. lar activity in vitro, given intracellular ATP ranges from 1 to 10 mM

LC20 phosphorylation within HS56-treated vessels was reduced (Beis and Newsholme, 1975).

to near baseline levels, with unphosphorylated and monophos-

phorylated LC20 predominating (Figure 5B), whereas no reduc- selective DAPK3 inhibition were instead manifested in contrac-

tion was observed within other treatment groups. tile intensity and LC20 phosphorylation. Maximum Cla-induced

Remarkable differences between Pim/DAPK3 inhibition and

Few differences in contraction rates were observed between force, expressed as percentage of K⁺-induced reference force,

inhibitors, all of which increased the time required to reach was signiﬁcantly reduced (>75%) by HS56, whereas no reduc-

50% of maximal Cla-induced contraction (t_{1/2 max}) (Figure 5C). tion was observed with other inhibitors. HS56 caused signiﬁcant

Concentration-dependent studies conﬁrmed that HS56, HS94, concentration-dependent force reduction at [HS56] >10 mM with

and HS148 acted similarly to delay force development (Fig- ꢀ3-fold reduction observed at [HS56] >75 mM. HS56 caused

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Figure 5. HS56 Delayed Force Onset, Decreased Contractile Force, and Reduced LC20 Phosphorylation in Excised Rat Caudal Arterial VSM

Tissues

(A) Representative calyculin A (Cla)-induced contractile traces following treatment with vehicle (DMSO) or inhibitor.

(B) Phos-tag SDS-PAGE western blot analysis of LC20 phosphorylation from non-phosphorylated (0P) to triphosphorylated (3P) following treatment with vehicle

(NH), Cla, or Cla + inhibitor.

(C) Effect of inhibitors on t_{1/2 max}, maximum force, and total phospho-LC20/total LC20 (determined by scanning densitometry quantitation) from Cla-stimulated

contractions.

(D) Titration curves derived from multiple contractile traces in the presence of Cla + inhibitor.

Mean SEM, n = 3. Signiﬁcantly different from Cla (ANOVA, Dunnett’s post hoc test, n > 3), *p < 0.002.

4-fold reduction in LC20 phosphorylation in the same tissues. phorylated state. Because all Pims lack a calmodulin regulatory

Similar trends were not evident with HS94 and HS148 at concen- domain, this mechanism of action must be Ca²⁺independent.

trations up to 100 mM. These results provided initial indication Indeed, ex vivo experiments showed that DAPK3 and Pim/

that HS56 modulates LC20 phosphorylation and VSM contrac- DAPK3 inhibitors affect neither kinetics nor intensity of supra-

tility and that Pim/DAPK3 inhibition affects VSM in a fundamen- maximal Ca²⁺(pCa4.5)-induced contractions of rat caudal arte-

tally different manner from selective DAPK3 inhibition.

rial strips. However, whether coincident Pim/DAPK3 inhibition

acts to decrease Ca²⁺sensitivity with a rightward shift in the

pCa-tension response curve remains to be determined (Figures

Mechanistic Insight into Pim Activity

We hypothesized that Pim kinases play a role in VSM contraction S6C and S6D).

by directly phosphorylating regulatory residues on LC20 (Thr18,

The coulombic surfaces of DAPK3 and Pim-1 (complexed with

Ser19) and/or MYPT1 (Thr697, Thr855), thereby suppressing PimTide) (Pogacic et al., 2007) showed acidic, negatively charged

MLCP activity and shifting the balance of myosin to the phos- peptide binding grooves traversing their respective C-terminal

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doi.org/10.1016/j.chembiol.2018.06.006

Figure 6. Pim Kinases Directly Phosphory-

late VSM Substrates

(A and B) Structural similarities between peptide

binding grooves (yellow box) of DAPK3 and

Pim-1. (A) DAPK3 coulombic surface with HS38

(gray sticks). (B) Coulombic surface of Pim-1 with

PimTide substrate (gray sticks) (PDB: 2C3I) (Po-

gacic et al., 2007); (À), acidic (red); (+), basic (blue),

neutral (white); black arrows indicate approximate

location of peptide phosphorylation site.

(C) Pim substrates (PimTide, Dundee Substrate)

and DAPK3 substrates (MYPT1, LC20) contain

basic residues N-terminal to their phosphorylation

sites (Ser*/Thr*).

(D) Phos-tag SDS-PAGE western blot depicting

phosphorylation of LC20 by MLCK and Pim

kinases.

(E) Phos-tag SDS-PAGE western blot depicting

phosphorylation of MYPT1 by Pim kinases and

regulators of VSM contractility (ROCK, DAPK3).

Inhibition by HS56, but not inactive analog HS182;

[Analog] = 50 mM.

The ability of Pims to directly

phosphorylate LC20 and MYPT1 was

investigated in vitro (Figures 6D and 6E,

a subset of Figure S7). Phosphorylation

of LC20 by Pim-1 was minor, compared

with MLCK, after 60 min. No evidence

of LC20 phosphorylation by Pim-2 was

observed. LC20 phosphorylation by

Pim-3 over 60 min was incomplete;

however, mono- and diphosphorylated

products were clearly visible. Although

relatively slow, Pim-3 activity likely con-

tributes to contraction and maintenance

of vascular tone.

All three Pims rapidly and robustly

phosphorylate MYPT1 at multiple sites

on time scales similar to, or faster than,

DAPK3 and ROCK. Evidence of phos-

phorylated products was clear after 30 s

and nearly complete conversion to mono-

phosphorylated MYPT1 was apparent af-

ter 0.5–5 min. Pim-2 produced primarily

mono- and diphosphorylated MYPT1

products while Pim-1 and Pim-3 pro-

duced tri- and tetraphosphorylated prod-

ucts. All Pim kinases were inhibited by

HS56, but not by inactive analog HS182.

catalytic lobes (Figures 6A and 6B). Basic substrates align within DAPK3 activity was also inhibited by HS56, while ROCK activity

these grooves with phosphorylation sites oriented toward the was unaffected. These results strongly suggest that Pim kinases

g-phosphate of ATP. DAPK3 displays high afﬁnity for basic sub- are regulators of VSM contractility via direct phosphorylation

strates within muscle, e.g., MYPT1 and LC20, and Pim kinases of MYPT1.

display similarly high afﬁnity for basic substrates of the general

form RXRHP(S*/T*)G (Pogacic et al., 2007), e.g., Dundee sub- HS56 Normalizes BP In Vivo

strate(Bain e t a l., 2003) and Pimtide. Sequencealignmentofthese Having established that HS56 modulates VSM contractility in

peptide substrates shows numerous basic residues N-terminal to tissues, we tested whether Pim/DAPK3 inhibition lowered

their phosphorylation sites (Figure 6C), which implies that Pim BP in spontaneously hypertensive renin transgene (RenTg)

kinases likely display afﬁnity for VSM substrates.

(Caron et al., 2002) and wild-type (WT) mice. Acute systemic

8

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Figure 7. HS56 Lowers BP in Spontaneously Hypertensive Mice

HR and BP data recorded during intravenous bolus delivery of vehicle (DMSO; n R 4), HS148 (10 mg/kg; n = 3), and HS56 (10, 20 mg/kg; n R 3) into anesthetized,

spontaneously hypertensive RenTg and WT mice.

(A) Representative SBP and HR tracings from single RenTg (top) and WT (bottom) mice. Black arrows indicate time of infusion. DMSO effect on HR (gray arrows)

caused reversible lowering of SBP.

(legend continued on next page)

Cell Chemical Biology 25, 1–13, September 20, 2018

9

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vasodilatory effects were observed with HS56, but not HS148. (15- to 35-fold less potent than HS56), and low micromolar to

Both HS56 and HS148 were formulated as DMSO solutions. no activity versus Pim-1 and Pim-2. Thus, HS94 and HS148

Intravenous infusion of HS56 into RenTG mice induced dose- are sufﬁciently selective for probing DAPK3 inhibition in the

dependent decreases in systolic blood pressure (DSPB = absence of Pim inhibition in tissues and animals.

À20 7 and À28 3 mmHg, at 10 and 20 mg/kg, respectively,

All Pim kinases phosphorylated MYPT1 in vitro (Figure 6E).

measured 2.2 min after infusion) without decreasing the heart HS56 substantially inhibited MYPT1 phosphorylation by the

rate (HR) (Figures 7A–7D). HS56 (20 mg/kg) lowered SBP Pims and DAPK3, but not ROCK. Although phosphorylation of

of RenTg mice to near WT levels (from ꢀ119 mmHg to LC20 by Pim-3 occurred slowly compared with MLCK, we

ꢀ91 mmHg) while HR increased slightly (from ꢀ412 bpm to reasoned that this activity was physiologically relevant based

ꢀ436 bpm), measured 2.2 min after infusion. HS56-associated on tissue studies. In VSM tissues, HS56, HS94, and HS148 de-

HR increases were likely compensatory effects caused by vaso- layed force-onset kinetics; however, insigniﬁcant differences

dilation. In WT mice, HS56 affected neither HR nor SBP at among their effects suggest that the role of Pim kinases is not

10 mg/kg and, at 20 mg/kg, decreased SBP from ꢀ99 mmHg yet kinetically discernible from that of DAPK3. Remarkably, se-

to ꢀ76 mmHg (DSBP = À23 5 mmHg) without changing HR. lective inhibition of DAPK3 alone (HS94, HS148) was insufﬁcient

Neither HR nor SBP of RenTg and WT mice were signiﬁcantly to reduce contractile force, while concurrent Pim/DAPK3 inhibi-

affected by HS148 (10 mg/kg).

tion by HS56 reduced maximum force and drove LC20 phos-

Interestingly, vehicle (DMSO) alone caused an immediate drop phorylation to near baseline levels (Figure 5). In these experi-

in HR and SBP that was profound, short-lived, and immediately ments, Cla inhibits MLCP and, in the absence of phosphatase

reversible (DMSO effect). This is likely due to jugular vein delivery activity, LC20 phosphorylation induced contraction. Pretreat-

of undiluted DMSO (30–60 mL) directly into the heart, causing ment with potent inhibitors of MYPT1 phosphorylation (e.g.,

transient slowing of HR, which lowered BP. Upon recovery, HR HS94, HS148) was sufﬁcient to lengthen t_{1/2 max}, but force even-

and BP returned to stable baseline levels (ꢀ0.5 min and tually reached a stable maximum due to persistent activities of

ꢀ1.5 min after infusion, respectively). As expected, the DMSO MLCK and other LC20 kinases. The ability of HS56 to reduce

effect was also observed after infusion of inhibitors; however, maximum force, by deduction, necessarily involves immediate

HS56 effects persisted for the duration of monitoring and were or upstream inhibition of LC20 phosphorylation.

readily distinguishable from DMSO effects. These results

Most notably, Pim/DAPK3 inhibition by HS56 restored near

suggest that the combined activity of DAPK3 and Pim kinases normal SBP in RenTg mice (DSBP = À28 3 mmHg; ꢀ23%

contributes to BP regulation and hypertension in vivo and that reduction) without diminishing HR, while DAPK3 inhibition by

the pathophysiology of hypertension can be acutely abrogated HS148 affected neither SBP nor HR (Figure 7). HS56 displayed

by HS56.

dose-dependent, rapid lowering of SBP that persisted for

several minutes; characteristics that are consistent with ideal

HE therapeutics (Tulman et al., 2012) and compare favorably

with, or surpass those of ROCK and P2X3 antagonists reported

DISCUSSION

Chronic VSM hypercontractility leads to hypertension, vascular in notable hypertension studies. ROCK inhibition by Y-27632

hypertrophy, and signiﬁcant cardiovascular disease. The contri- (IC₅₀= 800 nM) produced a 50- to 80-mmHg (20%–33%) drop

bution of Pim kinases to VSM contractility and pathogenesis of in BP in hypertensive mice (Uehata et al., 1997); however, slow

hypertension has been largely uncharacterized. Here we devel- onset (ꢀ1 hr), long duration of action (>7 hr), and off-target ef-

oped and characterized a set of molecular probes, which were fects (e.g., PRK2; IC₅₀= 600 nm) (Bain et al., 2007) diminish its

utilized to demonstrate that (1) Pims directly target VSM sub- therapeutic value for both chronic hypertension and HE. The

strates in vitro; (2) Pim/DAPK3 inhibition by HS56 critically mod- P2X3 receptor antagonist, AF-219, produced a 28-mmHg

ulates LC20 phosphorylation and contractility in tissues; and (ꢀ19%) drop in SBP in hypertensive rats, but this was accompa-

(3) HS56 regulates BP in vivo without affecting heart rate.

Based on molecular properties, kinome proﬁling, kinase as- dramatic contrast to HS56, which had little impact on the heart.

says, and ex vivo studies, HS56 satisﬁes the criteria for high- These studies provide mechanistic biological insight into the

quality chemical probes (Arrowsmith et al., 2015). In brief, these polypharmacology of Pims and DAPK3 in the context of VSM

include: (1) deﬁned mode of action; (2) selective: S(10)_HS56contraction; establishing Pim kinases as potentially critical ele-

nied by a signiﬁcant drop in HR (ꢀ10%) (Pijacka et al., 2016), in

=

0.017 from 468 assays; (3) synthetically available; (4) deﬁned ments of a multi-target engagement strategy for controlling

structurally inactive analog (HS182); and (5) demonstrated ef- hypertension. HS56 is key molecular probe for elucidating the ef-

fects in tissues at 1 mM. Based on our ﬁndings, HS56 is suitable fect of Pim/DAPK3 inhibition on VSM contractility and represents

for characterization of dual DAPK/Pim inhibition in VSM systems a signiﬁcant ﬁrst step in the development of next-generation

and hypertension.

antihypertensive medications. If HS56 or future generations of

HS94 and HS148 show a high degree of selectivity for DAPK3 Pim/DAPK3 inhibitors produce clinically predictable dose-

(2.5- to 3-fold more potent than HS56), low potency for Pim-3 dependent reduction in systemic BP without altering HR, this

(B) Changes in HR and SBP during infusions into RenTg (left) and WT (right) mice. Data points are binned averages of 1 s from every 5 s of data. Black arrows

indicate time of infusion and gray arrows the DMSO effect.

(C) Average change in HR at t = 2.5 min.

(D) Average change in SBP at t = 2.5 min.

Data points in (B) to (D) are mean SEM. Signiﬁcantly different from DMSO (two-way ANOVA), *p < 0.05, **p < 0.01, ***p < 0.001.

10 Cell Chemical Biology 25, 1–13, September 20, 2018

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doi.org/10.1016/j.chembiol.2018.06.006

for performing all mouse acute infusion studies and data annotation; Ingo

certainly would constitute a signiﬁcant advance in the molecular

Korndoerfer and the team at CRELUX (Martinsried, Germany) for crystallog-

management of acute hypertension, thereby reducing morbidity

raphy studies; Nathan Nicely at the Duke University Macromolecular X-ray

and mortality rates associated with the condition. We anticipate

Crystallography Shared Resource for crystallography expertise and PDB sub-

that chronic administration of HS56 may also mitigate and

mission; Michael Walsh (University of Calgary) for helpful discussions

reverse hypertrophic remodeling and chronic hypertension-

regarding VSM data; Kavita Pillai, Juliane Totzke, and Kirsten Overdahl for

related pathologies.

helpful conversations. This work was supported in part by a grant from the

Mandel foundation to T.A.J.H; the Canadian Institutes of Health Research

(CIHR MOP-97988; CIHR MOP-133543) and the AIHS/Pﬁzer Translational

Research Fund to J.A.M; the National Institute of Diabetes, Digestive and Kid-

ney Diseases (Duke O’Brien Center for Kidney Research, NIH award number

P30DK096493) to S.B.G.; and a grant from the Department of Veterans Affairs

to MAS (#IK2BX002240). All animal experiments were conducted according to

protocols approved by the institutional Animal Care and Use Committees for

the University of Calgary, Duke University School of Medicine, and

Durham VAMC.

SIGNIFICANCE

Investigations into underlying mechanisms governing VSM

contractility remain vitally important for developing molecu-

larly targeted antihypertensive medications. Here we

demonstrated that Pim kinases directly target VSM sub-

strates in vitro. Additionally, we developed a set of inhibitors

that were utilized as molecular probes to demonstrate that

dual Pim/DAPK3 inhibition by HS56 (1) critically modulates

LC20 phosphorylation and contractility in tissues and (2) reg-

ulates BP in vivo without affecting heart rate. These studies

provide mechanistic insight into the polypharmacology of

VSM contraction, establishing Pim kinases as potentially

critical elements of a multi-target engagement strategy for

controlling hypertension.

AUTHOR CONTRIBUTIONS

D.A.C. carried out all synthetic work, analog characterization, kinase assays,

data analysis, and wrote the paper with T.A.J.H. M.R.S. performed kinase as-

says, crystal structure analysis, and ﬁgure design with D.A.C. C.S. and J.A.M.

conceived and coordinated phosphorylation studies, VSM substrates, and all

excised VSM experiments. J.A.M. assisted with manuscript and ﬁgure prepa-

ration. C.R., L.T.A., and S.K. expressed and puriﬁed Pim kinases for enzymatic

and kinase assays. P.F.H. performed initial synthesis of HS38 and selected an-

alogs. S.B.G. and M.A.S. supervised acute infusion studies in RenTg and WT

mice and assisted in data analysis. T.A.J.H. is the principal investigator of this

project and, along with D.A.C., designed and conceived all studies described

here. All authors reviewed the data and approved the ﬁnal version of the

manuscript.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

DECLARATION OF INTERESTS

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Bacteria Culture

T.A.J.H. and D.A.C. are inventors on a related patent (US, 2017/0260191 A1,

WO/2016/049500). J.A.M. is co-founder of ArchBiopartners and a member

of its scientiﬁc advisory board.

B Tissue Preparation

Received: September 25, 2017

Revised: March 16, 2018

Accepted: June 20, 2018

Published: July 19, 2018

B Animal Models and Welfare

d METHOD DETAILS

B Human Protein Atlas and Expression Atlas

B Expression and Puriﬁcation of Kinases

B Preparation of Kinase Assay Substrates

B Kinase Assays

SUPPORTING CITATIONS

The following reference appears in the Supplemental Information: Ihara and

MacDonald, 2007.

B Tissue Preparation and Force Measurements

B LC20 Phosphorylation in Tissues

B In Vitro Phosphorylation Studies

B X-Ray Crystallography

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d QUANTIFICATION AND STATISTICAL ANALYSIS

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Cell Chemical Biology 25, 1–13, September 20, 2018 13

Please cite this article in press as: Carlson et al., Targeting Pim Kinases and DAPK3 to Control Hypertension, Cell Chemical Biology (2018), https://

doi.org/10.1016/j.chembiol.2018.06.006

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

Antibodies

SOURCE

IDENTIFIER

anti-MYL2

Santa Cruz Biotechnology

New England Peptide

Cat# sc-517414

anti-panMYPT1

Custom

anti-LC20

Santa Cruz Biotechnology

Cat# sc-48414; RRID:AB_2148042

Bacterial and Virus Strains

Escherichia coli BL21 (DE3)-R3

Structural Genomics Consortium,

University of Oxford

NA

(Strain-Damerell et al., 2014)

Escherichia coli BL21(DE3)pLysS

Chemicals, Peptides, and Recombinant Proteins

HS38

Promega

Cat# L1195

Own Production

N/A

(Carlson et al., 2013)

Analogs of HS38

MLCK

Own Production

(This Paper)

Justin MacDonald,

University of Calgary

Chicken Gizzard

(Ngai et al., 1984)

ROCKa (ROCK2; residues 2-543)

EMD Millipore

Cat# 14-338

N/A

PKA

Justin MacDonald,

University of Calgary

Bovine Heart

(Grassie et al., 2012)

Peptide substrate for DAPK3, KKKRQSRRSTQGVTL,

corresponding to Arg690 to Lys701 of MYPT1

University of Calgary

Peptide Services

Custom

(MacDonald et al., 2001a)

Peptide substrate for Pim Kinases, RSRHSSYPAGT

LC20

Biomatik

Custom

Justin MacDonald,

NCBI accession: NP_990609

University of Calgary

Chicken Gizzard

(Hathaway and Haeberle, 1983)

PreScission Protease for GST-tag removal

[g-³²P]-ATP

GE Healthcare

Perkin Elmer

BioBasic

Cat# 27084301

Cat# NEG035C005MC

Cat# AB0020

ATP disodium salt

Ni Sepharose Resin

GE Healthcare

EMD Millipore

FujiFilm Wako

Thermo Fisher

Sigma-Aldrich

Matrix Scientiﬁc

Sigma-Aldrich

Matrix Scientiﬁc

Alfa Aesar

Cat# 17-5268-01

Cat# 208851

Calyculin A

Phos-Tag SDS-PAGE

Cat# AAL-107

Cat# 34095

West Femto enhanced chemiluminescence reagent

TEV Protease

Cat# T4455

Ethyl-2-cyano-3-ethoxyacrylate

phenylhydrazine

Cat# E28005

Cat# P26252

2-chlorophenylhydrazine hydrochloride

3-chlorophenylhydrazine hydrochloride

4-chlorophenylhydrazine hydrochloride

isopropylhydrazine hydrochloride

o-tolylhydrazine hydrochloride

m-tolylhydrazine hydrochloride

Cat# 109509

Cat# 075249

Cat# C65807

Cat# 007266

Cat# A15767

Acros

Cat# 139150010

(Continued on next page)

e1 Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018

Please cite this article in press as: Carlson et al., Targeting Pim Kinases and DAPK3 to Control Hypertension, Cell Chemical Biology (2018), https://

doi.org/10.1016/j.chembiol.2018.06.006

Continued

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Cat# 021395

Cat# 021131

Cat# AK-39869

Cat# 453471

Cat# 003925

Cat# 005466

Cat# B20326

Cat# 261653

Cat# C19651

Cat# 167185

Cat# 17457

3-methoxyphenylhydrazine hydrochloride

4-methoxyphenylhydrazine hydrochloride

3-cyanophenylhydrazine hydrochloride

4-cyanophenylhydrazine hydrochloride

3-ﬂuorophenylhydrazine hydrochloride

3-(triﬂuoromethyl)phenylhydrazine hydrochloride

4-hydrazinobenzoic acid hydrochloride

benzoyl isothiocyanate

Matrix Scientiﬁc

Ark Pharm, Inc

Sigma-Aldrich

Matrix Scientiﬁc

Alfa Aesar

Sigma-Aldrich

chloroacetonitrile

methyl 2-bromopropionate

methyl 2-bromoisobutyrate

methyl 2-bromobutyrate

Cat# 237310

Cat# 138630

Cat# 201170

Cat# 241067

2,4-dinitrochlorobenzene

phosphorus(V) oxychloride

2-methoxyethylamine

Critical Commercial Assays

Active Site-Directed Competition Binding Assay

against 468 Kinases

Euriﬁns DiscoverX Corp

KINOMEscan Scanmax Screen

Ionotropic and G Protein-Coupled

Receptor Assay

PDSP, Dept. of Pharmacology,

UNC Chapel Hill

Primary Binding (% Inhibition)

Screen

https://pdspdb.unc.edu/pdspWeb/

Deposited Data

Human Protein Atlas

https://www.proteinatlas.org/

(Uhlen et al., 2015)

N/A

Expression Atlas European Bioinformatics Institute

https://www.ebi.ac.uk/gxa/home

(Petryszak et al., 2016)

Crystal Structure of DAPK3 bound to HS38

Crystal Structure of DAPK3 used as search model

Crystal Structure of Pim-1 bound to AMPPNP

Crystal Structure of Pim-1 bound to PimTide

Crystal Structure of DAPK1 bound to AMPPNP

Experimental Models: Organisms/Strains

Sprague-Dawley rats

(This Paper)

PDB: 5VJA

PDB: 1P4F

PDB: 1XR1

PDB: 2C3I

PDB: 3F5U

(Huber et al., 2012)

(Qian et al., 2005)

(Pogacic et al., 2007)

(McNamara et al., 2009)

Charles River Laboratories,

Crl:SD

Strain code:400

Renin Transgene (RenTg) Mice and Normotensive

(WT) littermates

Duke Animal Models Core

(Caron et al., 2002)

Jackson Lab Cat# 007853

Also available from Jackson Laboratory

Oligonucleotides

Bacterial expression vector pGEX-6P1

Recombinant DNA

GE Healthcare

Cat# 28954648

cDNA encoding DAPK3 (a.a. 1-320)

Genome Systems Inc

(Borman et al., 2002)

Cat# AI660136

NCBI accession: NP_001339

cDNA encoding Pim-1 (aa 1-312)

cDNA encoding Pim-2 (aa 1-311)

cDNA encoding Pim-3 (aa 1-326)

cDNA encoding full-length chicken MYPT1

Structural Genomics Consortium,

University of Oxford

Addgene Cat# 39132

UniProtKB: P11309

Structural Genomics Consortium,

University of Oxford

Addgene Cat# 38867

UniProtKB: Q9P1W9

Structural Genomics Consortium,

University of Oxford

UniProtKB: Q86V86

Dr. David Hartshorne,

University of Arizona

PPP1R12A, NCBI

accession: NP_990454

(Continued on next page)

Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018 e2

Please cite this article in press as: Carlson et al., Targeting Pim Kinases and DAPK3 to Control Hypertension, Cell Chemical Biology (2018), https://

doi.org/10.1016/j.chembiol.2018.06.006

Continued

REAGENT or RESOURCE

Software and Algorithms

Graphpad Prism 7

SOURCE

IDENTIFIER

Graphpad Software

GE Healthcare

FujiFilm

N/A

ImageQuant TL software

Chemiluminescence Analysis Multi-Gauge

version 3.0

Crystallographic Data Reduction

Crystallographic Data Scaling

Crystallographic Data Phasing

UCSF Chimera

MOSFLM

SCALA

N/A

AMoRE

https://www.cgl.ucsf.edu/chimera/

(Pettersen et al., 2004)

PoseView, ZBH Center for Bioinformatics

https://poseview.zbh.uni-amburg.de/

(Stierand and Rarey, 2010)

N/A

Powerlab Data Acquisition System with Labchart

Other

ADInstruments

P81 ion exchange cellulose chromatography paper

PVDF membrane

Whatman

Sigma

Cat# 3698325

Cat# 3010040001

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact,

Timothy Haystead (https://www.cell.com/action/showMethods?pii=S2451-9456%2817%2930269-6timothy.haystead@duke.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Bacteria Culture

Escherichia coli BL21 (DE3)-R3 cells (Strain-Damerell et al., 2014) and Escherichia coli BL21(DE3)pLysS (Sutherland et al., 2016) were

cultured in either terriﬁc broth (TB) or Luria-Bertami (LB) medium (supplemented with 50 mg/mL of appropriate antibiotic) at 37^ꢁC.

Tissue Preparation

Tissues were harvested from male Sprague-Dawley rats (ꢀ300 g, age 10-12 weeks) (Charles River Laboratories, Crl:SD Strain

code:400) that had been housed, anesthetized, and euthanized as previously described (MacDonald et al., 2016; Moffat et al.,

2011; Sutherland et al., 2016) according to protocols approved by the University of Calgary Animal Care and Use Committee consis-

tent with the standards of the Canadian Council on Animal Care.

Animal Models and Welfare

Hypertensive adult male mice (age 4-6 months) bearing a transgene expressing renin (RenTg) and normotensive (WT) littermates

(Duke Animal Models Core, also available fromThe Jackson Laboratory, Cat# 007853) were bred for use in experiments as previously

described (Caron et al., 2002). All animal experiments were conducted in compliance with institutional policies and appropriate

regulations and were approved by the institutional animal care and use committees for Duke University School of Medicine

(Duke IACUC A129-16-06) and Durham VAMC (1506-002).

METHOD DETAILS

Human Protein Atlas and Expression Atlas

A bioinformatic analysis of the Human Protein Atlas database (HPA) (Uhlen et al., 2015) using search terms (Pim1, Pim2, and Pim3)

provided graphical representations of HPA data in the form of a Tissue Atlas (Figure S1A). Each bar on the resulting Tissue Atlas

represents the highest expression score found in a particular group of tissues. Expression scores were automatically calculated

based on archived datasets within the HPA to represent (i) Pim kinase RNA expression from RNA-seq results, reported as number

of transcripts per million (TPM) and (ii) Pim kinase protein expression score based on a best estimate of the "true" protein expression

from knowledge-based annotation of the HPA database. Detailed institutional methods for HPA data collection, analysis, and scoring

can be found online at https://www.proteinatlas.org/about/assays+annotation#ihk. Representative results and Tissue Atlas for each

Pim kinase can be found online at:

e3 Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018

Please cite this article in press as: Carlson et al., Targeting Pim Kinases and DAPK3 to Control Hypertension, Cell Chemical Biology (2018), https://

doi.org/10.1016/j.chembiol.2018.06.006

http://www.proteinatlas.org/ENSG00000137193-PIM1/tissue

http://www.proteinatlas.org/ENSG00000102096-PIM2/tissue

http://www.proteinatlas.org/ENSG00000198355-PIM3/tissue

A second bioinformatic analysis of baseline RNA expression for Pim kinases in human tissues was conducted using the Expression

Atlas (Petryszak et al., 2016) provided by the European Bioinformatics Institute, found online at https://www.ebi.ac.uk/gxa/home,

using a Boolean combination of search terms (Pim# AND Homo sapiens; where # = 1, 2, or 3). Data from individual searches

were visualized, downloaded, and depicted as heatmaps representing Pim gene expression values, which were converted into a

color-scale image, providing a visual representation of gene expression levels across different biological locations and different in-

dependent experiments (Figure S1B). Data representing RNA expression for Pim-1, Pim-2, and Pim-3 within smooth muscle tissues

was found in datasets entitled 68 RANTOM5 and 32 Uhlen’s Lab.

Expression and Puriﬁcation of Kinases

DAPK3 (ZIPK)

Human DAPK3 sequence (NCBI accession: NP_001339) was obtained as cDNA clone (Genome Systems Inc., St Louis, MO,

Cat# AI660136) (Borman et al., 2002) and then subcloned into bacterial expression vector pGEX-6P1 (GE Healthcare,

Cat# 28954648). The catalytic domain of constitutively-active DAPK3 consisting of N-terminal residues 1-320 was expressed in

E. coli (BL21(DE3)pLysS (Promega, Cat# L1195) as a recombinant GST-fusion (GST-DAPK3). Bacteria were grown in standard

Luria-Broth and protein production was induced by addition of 0.1 mM IPTG for 4 hours at 37^ꢁC. Bacteria were collect by

centrifugation and resuspended in Buffer A, frozen at - 80^ꢁC and then lysed at room temperature with lysozyme (5 mg/25 ml culture).

After centrifugation at 10,000 3 g for 30 min, the supernatant was removed and GST-DAPK3 (1-320) was captured with glutathione-

Sepharose afﬁnity chromatography. After extensive washing (Buffer A + 0.5M NaCl), the bead slurry was incubated with PreScission

Protease for GST-tag removal (GE Healthcare, Cat# 27084301) for 16 hours at 4^ꢁC. Eluted fractions were recovered and analyzed by

SDS-PAGE to identify pure DAPK3(1-320) material.

Pim Kinases

cDNAs encoding human full length Pim kinases (Pim-1,-2,-3) were obtained from synthetic sources and used as templates to amplify

kinase domain-containing sequences and further sub-cloned into different expression vectors, using ligation independent cloning

(Strain-Damerell et al., 2014). Pim-1 (aa 1-312) was cloned into PLIC-SGC1 [pET expression vector with His₆tag in a 23 aa-N-terminal

fusion peptide, with TEV protease cleavage site, (Amp⁺)] and co-expressed with l-phosphatase in Escherichia coli BL21 (DE3)-R3

cells. Pim-2 (aa 1-311) and Pim-3 (aa 1-326) were cloned into pNIC28-Bsa4 [pET expression vector with His₆tag in a 22 aa-N-terminal

fusion peptide, with TEV protease cleavage site, (Kan⁺)] and co-expressed with l-phosphatase in Escherichia coli BL21 (DE3)-R3

cells. Transformed cells were initially cultured (from an overnight pre-culture) in either terriﬁc broth (TB) medium (supplemented

with 50 mg/mL of appropriate antibiotic) to OD₆₀₀of ꢀ1.6 at 37^ꢁC, followed by additional growth while cooling to 18^ꢁC to an

OD₆₀₀of ꢀ3 before induction with 0.5 mM IPTG overnight, or cultured in Luria-Bertami (LB) medium to OD₆₀₀of ꢀ0.4 at 37^ꢁC,

followed by additional growth while cooling to 18^ꢁC to an OD₆₀₀of ꢀ0.7 before induction with 0.5 mM IPTG overnight. Cells were

harvested by centrifugation (JLA 8,100 rotor Beckman Coulter, Avanti J-20 XP centrifuge) and were frozen at -20^ꢁC. Cells expressing

His₆-tagged proteins were re-suspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5% glycerol and

0.5 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) in the presence of protease inhibitors cocktail (1 mL/mL) and lysed

by sonication using a 750 W Sonics Vibra-Cell sonicator, with amplitude set to 35 %, with bursts of 5 sec on-10 sec off, for 5 minutes,

on ice. PEI (polyethyleneimine) was added to a ﬁnal concentration of 0.15 % and lysates were transferred to centrifuge tubes and

centrifuged at 53,0003g using a JA-25.50 rotor, for at least 45 minutes, at 4^ꢁC. After centrifugation, the clariﬁed supernatant was

passed through a gravity column of 5 mL Ni-Sepharose resin, IMAC (GE Healthcare, Cat# 17-5268-01), previously equilibrated in lysis

buffer. The resin was ﬁrst washed with 50 mL of lysis buffer containing 1 M NaCl and 30 mM imidazole, then with 25 mL of Lysis Buffer

containing 100 mM imidazole and ﬁnally the protein was eluted with 25 mL of Lysis Buffer containing 300 mM imidazole. The eluted

proteins were collected and treated overnight with TEV (Tobacco Etch Virus) protease (Sigma-Aldrich, Cat# T4455) at 40^ꢁC to remove

the N-terminal tag. Digested proteins were loaded onto a nickel column again to remove the cleaved hexa-histidine expression tag

protease used. The ﬂow-through containing the cleaved proteins was collected and concentrated to 5 mL (Amicon) and injected onto

a Superdex 75 or 200 (16/60) gel ﬁltration column on an AKTA system (GE Healthcare) pre-equilibrated into GF Buffer (50 mM HEPES

pH7.5, 300 mM NaCl, 5% glycerol, and 0.5 mM TCEP). The resulting pure protein was stored at À80^ꢁC in 50 mM HEPES, pH 7.5,

300 mM NaCl, 0.5 mM TCEP and 5% glycerol. The correct mass and purity for all protein constructs was conﬁrmed by an Agilent

1100 Series LC/MSD TOF (Agilent Technologies Inc., Palo Alto, CA).

MLCK

MLCK was puriﬁed from chicken gizzard as previously described (Ngai et al., 1984).

ROCKa

Constitutively-active N-terminal His₆-tagged and N-terminal HA-tagged, recombinant rat ROCKa (ROCK2; residues 2-543;

67.2 kDa), expressed by baculovirus in Sf21 insect cells, was purchased from EMD Millipore (catalog number 14-338).

PKA

PKA was puriﬁed from bovine heart as previously described (Grassie et al., 2012).

Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018 e4

Please cite this article in press as: Carlson et al., Targeting Pim Kinases and DAPK3 to Control Hypertension, Cell Chemical Biology (2018), https://

doi.org/10.1016/j.chembiol.2018.06.006

Preparation of Kinase Assay Substrates

Peptide Substrate for DAPK3

MYPT1 substrate peptide (KKKRQSRRSTQGVTL), corresponding to Arg690 to Lys701 of MYPT1, was synthesized by University

of Calgary Peptide Services (Calgary, Alberta), conﬁrmed by amino acid analysis, and shown to be 95% pure by analytical high-

performance liquid chromatography. (MacDonald et al., 2001a)

Peptide Substrate for Pim Kinases

RSRHSSYPAGT was custom synthesized by Biomatik (Wilmington, Delaware, USA).

LC20

Smooth muscle myosin regulatory light chains (LC20, also termed MLC2; NCBI accession: NP_990609) were puriﬁed from chicken

gizzard as described (Hathaway and Haeberle, 1983). Brieﬂy, tissue was homogenized in a blender in buffer containing MOPS,

pH 6.8, 20 mM KCl, 1 mM dithiothreitol, 1 mM EGTA, 1 mM MgCl₂, and 0.2% Triton X- 100 (Buffer A plus Triton). Subsequent

separation of whole myosin was achieved by a series of centrifugations (12,000 3 g) and resuspensions in Buffer A plus Triton. Pellets

from the ﬁnal centrifugation were solubilized by sonication in buffer containing 20 mM MOPS and 5 mM DTT, pH 7.0. This solution

was adjusted to contain ﬁnal concentrations of urea (8 M), guanidine HCl (1 M), and SDS (0.05%) at pH 8.0. Essential and regulatory

myosin light chains (LC17 and LC20, respectively) were enriched in solution by the addition of absolute ethanol at room temperature,

followed by centrifugation of unwanted precipitate at 12,000 3 g. The supernatant was dialyzed against buffer containing 5 mM

ammonium bicarbonate and 0.4 mM DTT. Following dialysis, the solution was adjusted to contain 20 mM MOPS, 0.6 M NaCl,

1 mM EDTA, 1 mM DTT, pH 7.5 (equilibration buffer) and loaded onto a column of phenyl-Sepharose. After washing with equilibration

buffer, LC20 was isolated from LC17 and other protein contaminants by elution with deionized water containing 1 mM DTT. Eluted

fractions were recovered and analyzed by SDS-PAGE.

MYPT1

Full-length chicken MYPT1 sequence (PPP1R12A, NCBI accession no. NP_990454) was generously provided by Dr. David

Hartshorne (University of Arizona). MYPT1 was sub-cloned into bacterial expression vector pGEX-6P1 (GE Healthcare,

Cat# 28954648) and transformed into E. coli strain BL21(DE3)pLysS (Promega, Cat# L1195). The recombinant GST-tagged

MYPT1 was puriﬁed as previously described (Sutherland et al., 2016). Brieﬂy, GST-MYPT1 protein production was induced in

E. coli BL21(DE3)pLysS by addition of IPTG for 4 hours at 37^ꢁC. Bacteria were collect by centrifugation and resuspended

in 30 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 0.2 mM DTT with protease inhibitors (Buffer A), frozen at -

80^ꢁC and then lysed at room temperature with lysozyme (5 mg/25 ml culture). After centrifugation at 10,000 3 g for 30 min, the

supernatant was removed, diluted 1:3 with Buffer A, and loaded onto an SP-Sepharose column. After development with a 0.15

to 0.5 M NaCl gradient, fractions containing GST-MYPT1 were pooled and captured with glutathione-Sepharose afﬁnity chromatog-

raphy. After extensive washing (Buffer A + 0.5 M NaCl), the bead slurry was incubated with PreScission Protease for GST-tag removal

(GE Healthcare, Cat# 27084301) at 4^ꢁC for 3 days. Eluted fractions were recovered and analyzed by SDS-PAGE to identify full-length

MYPT1 material. The MYPT1 protein was conﬁrmed to be free of kinase activity by an in vitro phosphorylation assay performed in the

absence of exogenous kinase.

Kinase Assays

General Protocol

Analogs were formulated as 10 mM solutions in DMSO and assayed against protein kinases using radiolabeled ATP in a protocol

adapted from the International Center for Kinase Proﬁling (Dundee, UK) (Hastie et al., 2006). Protein kinase, dissolved in 25 mM

HEPES pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol (HEPES-Mg buffer), was added to a solution of peptide substrate, ATP, analog,

and DMSO in HEPES-Mg buffer. The ﬁnal reaction volume of 40 mL contained 50 - 350 ng kinase, 140 - 300 mM peptide substrate,

5 - 50 mM ATP (Bio Basic, Markham, ON, Canada, Cat# AB0020) with 5 - 20 mCi of [g-³²P]-ATP (Perkin Elmer, Waltham, MA, USA,

Cat# NEG035C005MC), 10 mM analog (initial screen) or 0.001 mM – 100 mM analog (titration), and 2.5 % DMSO. For exact compo-

sitions, see below. After 10 min, the reaction was terminated by addition of concentrated H₃PO₄(10 mL). A 5 mL aliquot of each mixture

was spotted onto P81 ion exchange cellulose chromatography paper (Whatman Cat# 3698325), washed 33 with 200 mM H₃PO₄and

then 13 with acetone. After thorough drying, the papers were placed in uncapped scintillation vials and Cherenkov counted for

0.5 min using tritium isotope detection (Beckman LS6500 Scintillation Counter).

DAPK3 Kinase Assay

The ﬁnal reaction contained 52 ng GST-DAPK3, 140 mM MYPT1 peptide substrate, 50 mM ATP with 5 - 20 mCi of [g-³²P]-ATP, 10 mM or

0.001 mM – 100 mM analog, and 2.5 % DMSO in HEPES-Mg buffer.

Pim-1 Kinase Assay

The ﬁnal reaction contained 100 ng Pim-1, 300 mM Pim peptide substrate, 50 mM ATP with 5 - 20 mCi of [g-³²P]-ATP, 10 mM or

0.001 mM – 100 mM analog, and 2.5 % DMSO in HEPES-Mg buffer.

Pim-2 Kinase Assay

The ﬁnal reaction contained 350 ng Pim-2, 300 mM Pim peptide substrate, 5 mM ATP with 5 - 20 mCi of [g-³²P]-ATP, 10 mM or 0.001 mM

– 100 mM analog, and 2.5 % DMSO in HEPES-Mg buffer.

Pim-3 Kinase Assay

The ﬁnal reaction contained 150 ng Pim-3, 300 mM Pim peptide substrate, 50 mM ATP with 5 - 20 mCi of [g-³²P]-ATP, 10 mM or

0.001 mM – 100 mM analog, and 2.5 % DMSO in HEPES-Mg buffer.

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Determination of DAPK3 K_Mfor ATP

Using a variation of the kinase assay general protocol, GST-DAPK3 with saturating amounts of peptide substrate was assayed

against several concentrations of ATP. A stock solution of 4 mM ATP, containing 550 mCi/mL of [g-³²P]-ATP, was serially diluted

with HEPES-Mg buffer and aliquoted into the reactions. Experimental reactions: ﬁnal volumes of 40 mL contained 52 ng GST-

DAPK3, 140 mM MYPT1 peptide substrate, and ATP ranging from 1 mM – 500 mM with 0.006 – 3 mCi of [g-³²P]-ATP. Negative control:

each experiment reaction was repeated without GST-DAPK3, and the counts per minute were subtracted to remove background

signal. Data was analyzed by non-linear curve ﬁtting (Graphpad Prism 7) and Lineweaver-Burk double reciprocal plot (Figure S4F).

IC₅₀and K_iValues

IC₅₀values were determined from titration curves (Figure S4) using log(inhibitor) vs. response - Variable slope (four parameters)

(Graphpad Prism 7). The IC₅₀for HS56 titrated against Pim-2 was estimated from curve ﬁtting X-min = -3 (R²= 0.901). IC₅₀values

were converted to inhibition constants (K_i) using the Cheng Prusoff equation (Cheng and Prusoff, 1973) (Figure S4G). Previously

reported Michaelis constants (K_M) of Pim kinases for ATP were used; 160 mM (Pim-1), 4 mM (Pim-2), and 40 mM (Pim-3) (Burger

et al., 2013). For GST-DAPK3, the K_Mfor ATP was determined experimentally to be 20 mM (Figure S4E).

Tissue Preparation and Force Measurements

Caudal arteries were removed from male Sprague-Dawley rats (ꢀ300 g) that had been anesthetized and euthanized according to

protocols approved by the University of Calgary Animal Care and Use Committee consistent with the standards of the Canadian

Council on Animal Care. The arteries were cleaned of excess adventitia, denuded of endothelium, and cut into helical strips

(1.5 mm 3 6 mm). Muscle strips were mounted on a Grass isometric force transducer (FT03C) and force was recorded as previously

described (MacDonald et al., 2016; Moffat et al., 2011; Sutherland et al., 2016). Native tissues were maintained in HEPES-buffered

Tyrode’s (H-T) solution containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5.6 mM glucose, and 10 mM HEPES,

pH 7.4. Prior to induction, baseline contractile force was identiﬁed with application of H-T buffer containing 87 mM KCl and then

washed and pre-incubated with vehicle (DMSO), or inhibitor (HS38, HS56, HS94, HS148, HS182) at concentrations of 0-100 mM.

Contraction was induced with Calyculin A (Cla, 0.5 mM) (EMD Millipore, Cat# 208851) in H-T solution. Skinned (demembranated) tis-

sues were generated by incubation with 1% (v/v) Triton X-100 as described (Sutherland et al., 2016). Skinned tissues were washed

(3 3 5 min each) in pCa 9 solution (4 mM K₂EGTA, 5.83 mM MgCl₂, 0.5 mM DTT, 20 mM TES, pH 6.9, and an ATP regenerating system

composed of 3.9 mM Na₂ATP, 7.56 mM potassium propionate, 16.2 mM phosphocreatine, and 30 U/mL creatine kinase) and then

Ca²⁺-dependent contraction was induced with pCa 4.5 solution (4 mM CaEGTA, 5.66 mM MgCl₂, 0.5 mM DTT, 20 mM TES, pH 6.9,

and the ATP-regenerating system). Muscle strips were ﬂash frozen in 10% (w/v) trichloroacetic acid, 10 mM dithiothreitol in acetone,

followed by 3 3 10 second washes in 10 mM DTT in acetone. Tissues were then lyophilized overnight prior to protein extraction.

LC20 Phosphorylation in Tissues

LC20 phosphorylation in excised smooth muscle strips was quantiﬁed as previously described (MacDonald et al., 2016). Brieﬂy, rat

caudal artery VSM proteins were extracted from lyophilized tissues in 1 mL of SDS-gel sample buffer with constant shaking for 16

hours at 4^ꢁC prior protein resolution by Phos-Tag SDS-PAGE using 12.5% acrylamide and 50 mM Phos-Tag reagent (FujiFilm

Wako; Cat# AAL-107). Following transfer to PVDF membrane (Roche Life Science, Quebec, Canada), proteins were ﬁxed by incu-

bation for 20 minutes with 0.5% glutaraldehyde in phosphate-buffered saline and non-speciﬁc binding sites were blocked with

5% (w/v) nonfat dry milk. Membranes were incubated overnight with anti-LC20 (Santa Cruz Biotechnology, Cat# sc-48414, Santa

Cruz, CA) and then incubated for 1 hour with horseradish peroxidase–conjugated secondary antibody (Chemicon, Temecula, CA).

All western blots were visualized with West Femto enhanced chemiluminescence reagent (Thermo Fisher, Cat# 34095) using a

LAS4000 Imaging Station (GE Healthcare), ensuring that the representative signal occurred in the linear range. Quantiﬁcation was

performed by densitometry with ImageQuant TL software (GE Healthcare). LC20 phosphorylation stoichiometry was calculated

as follows: mol P_i/mol LC20 = [LC20-1P + (2 3 LC20-2P)] / [LC20-0P + LC20-1P + LC20-2P].

In Vitro Phosphorylation Studies

Reactions were performed at 30^ꢁC in a ﬁnal volume of 140 mL, initiated by addition of ATP (ﬁnal [ATP] = 0.5 mM), and terminated at the

indicated time by addition of SDS-PAGE sample buffer and boiling. After termination, samples were further diluted 1:10 and run on

Phos-Tag SDS-PAGE gels as described above. Experiments were repeated in the presence of inhibitor (50 mM) as indicated.

Phosphorylation of LC20 by MLCK

MLCK reactions contained 10 mg/mL LC20, 2 mg/mL MLCK, 25 mM HEPES pH 7.4, 10 mM MgCl₂, puriﬁed bovine calmodulin

(10 mg/mL), and 0.1 mM CaCl₂.

Phosphorylation of LC20 by Pim Kinases

Reactions contained 10 mg/mL LC20, 2 mg/mL Pim kinase, 25 mM HEPES pH 7.4, and 10 mM MgCl₂.

Phosphorylation of MYPT1 by ROCK

25 mM TRIS-HCl pH 7.5, 5 mM MgCl₂, 5 mM EGTA, 0.895 ng/mL ROCK, 20 mg/mL MYPT1, 0.5 mM ATP. Reactions were repeated

with HS182 and 50 mM HS56.

Phosphorylation of MYPT1 by Pim-1

25 mM HEPES pH 7.4, 10 mM MgCl₂, 2 mg/mL PIM1, 20 mg/mL MYPT1, 0.5 mM ATP. Reactions were repeated with HS182

and HS56.

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Phosphorylation of MYPT1 by Pim-2

25 mM HEPES pH 7.4, 10 mM MgCl₂, 2.8 mg/mL PIM1, 20 mg/mL MYPT1, 0.5 mM ATP. Reactions were repeated with HS182

and HS56.

Phosphorylation of MYPT1 by Pim-3

25 mM HEPES pH 7.4, 10 mM MgCl₂, 3 mg/mL PIM1, 20 mg/mL MYPT1, 0.5 mM ATP. Reactions were repeated with HS182

and HS56.

Phosphorylation of MYPT1 by DAPK3

25 mM HEPES pH 7.4, 10 mM MgCl₂, 2.5 mg/mL GST-DAPK3, 20 mg/mL MYPT1, 0.5 mM ATP. Reactions were repeated with HS182,

HS56, HS38, and HS94.

Phosphorylation of MYPT1 by PKA

25 mM TRIS-HCl pH 7.5, 5 mM MgCl₂, 5 mM EGTA, 1 mg/mL PKA, 20 mg/mL MYPT1, 0.5 mM ATP.

Western Blotting

Phosphorylation of LC20 (from puriﬁed chicken gizzard) by MLCK or PIM kinases was resolved using 12.5% acrylamide and 50 mM

Phos-Tag reagent (FujiFilm Wako; Cat# AAL-107) as described above. MYPT1 phosphorylation was quantiﬁed on optimized Phos-

Tag SDS-PAGE gels as previously described (Sutherland et al., 2016). Proteins were transferred to nitrocellulose membranes (0.2 mm)

overnight at 28 V and 4^ꢁC in 25 mM TRIS-HCl, pH 7.5, 192 mM glycine, and 10% (v/v) methanol. After blocking with 5% (w/v) nonfat

dry milk, membranes were incubated with primary antibody (MYL2 Antibody, Cat# sc-517414, Santa Cruz Biotechnology, Santa

Cruz, CA; anti-panMYPT1, custom made peptide-directed antibody, New England Peptide, Gardner, MA; 1:10,000 dilution) in

TBST for 2 h. Chemiluminescence signal detection using the SuperSignal West Femto reagent (Thermo Fisher, Cat# 34095) was

used following incubation with HRP-coupled secondary antibody (1:5,000 dilution). The emitted light was detected and quantiﬁed

with a chemiluminescence imaging analyzer (LAS3000mini, Fujiﬁlm), and images were analyzed with Multi-Gauge version 3.0

software.

X-Ray Crystallography

Expression, Puriﬁcation, and Crystallization

Expression and puriﬁcation of the DAPK3 catalytic domain for crystallographic studies was performed using proprietary methods by

Crelux GmbH (Martinsried, Germany). Brieﬂy, human DAPK3^Val9-Gly288was expressed as a fusion protein with TEV (tobacco etch

virus protease) cleavable N-terminal His₆afﬁnity tag in Escherichia coli strain BL21 (DE3). The fusion protein was puriﬁed by Ni afﬁnity

chromatography and His₆-TEV domain was removed by treatment with TEV protease. DAPK3 was isolated for crystallization studies

by size exclusion chromatography. Crystals of DAPK3 in complex with HS38 were obtained using hanging drop vapor diffusion

setups: 0.7 mL of DAPK3 (9.0 mg/mL in 50 mM HEPES, 500 mM NaCl, 5 mM DTT, 5 % glycerol, pH 7.5), pre-incubated with

5.4-fold molar excess of HS38 (150 mM in DMSO) for 2 h, mixed with 0.7 mL of reservoir solution (1.9 M sodium malonate,

pH 6.8) and equilibrated at 4^ꢁC over 400 mL of reservoir solution. Crystals appeared within 1 day and grew over 3-4 days to full size.

Data Collection

˚

A complete 2.5 A resolution data set of a DAPK3-HS38 crystal was collected at the ESRF synchrotron radiation source, id29,

˚

(Grenoble, France) using a Pilatus 6M (Dectris) detector at 0.97625 A wavelength. Data collection and reﬁnement statistics are listed

in Table S2. Data reduction was carried out with MOSFLM, and scaling with SCALA. Phasing was carried out using AMoRE.

Structure Determination and Reﬁnement

Initial structure determination was carried out by molecular replacement using a published DAPK3 structure as search model (PDB:

1P4F) (Huber et al., 2012). Alternating manual re-building and reﬁnement with REFMAC5 resulted in the ﬁnal model. Chains A, B, C,

and D are coincident with each other. Residues 49 to 53 in chain A, residues 47 to 53 in chain B, residues 52 to 54, 105 to 106 and 150

to 151 in chain C as well as residues 51 to 55, 104 to 106 and 150 to 151 in chain D could not be modelled due to ﬂexibility. The ﬁnal

model allowed for unambiguous placement of HS38.

Crystallographic Data Analysis

Molecular graphics were produced using the UCSF Chimera (Pettersen et al., 2004) package from the Computer Graphics Labora-

tory, University of California, San Francisco (supported by NIH P41 RR-01081). H-bonding was analyzed using FindHBond. Structural

overlays were created using MatchMaker. Analysis and 2D depiction of non-covalent protein-ligand interactions was performed

using PoseView (Stierand and Rarey, 2010) (ZBH Center for Bioinformatics, University of Hamburg, Germany).

Acute Infusion Studies in Mice

Acute systemic vasodilatory effects of HS148 and HS56 were examined in hypertensive RenTg mice and normotensive WT

littermates under 2% isoﬂurane anesthesia (Duke Animal Models Core) (Caron et al., 2002). A catheter (PE-50) was inserted into

the left jugular vein for the administration of basal ﬂuids and test compounds. A second catheter, pulled down PE-50 attached

to a pressure transducer (MLT844, ADInstruments, Colorado Springs, CO), was placed into the left carotid artery. Intra-arterial

BP was continuously recorded through the carotid catheter using the PowerLab data acquisition system and LabChart software

(ADInstruments, Colorado Springs, CO). HS148, HS56 or vehicle (DMSO) were injected intravenously into the internal jugular vein

at a volume of 1 ml/g body weight (25-30 ml total volume depending on weight) followed immediately by an equivalent bolus of vehicle

(total infusion 2 ml/g body weight, 50-60 ml). Vehicle-only mice received two identical vehicle infusions in rapid succession so that mice

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were exposed to the same volumes. HS148, HS56 and vehicle were examined in separate mice to minimize residual effects of the

drug. Intra-carotid BP data were analyzed by averaging 1 second of data every 5 seconds.

Chemical Synthesis

Abbreviations

DMF N,N-Dimethylformamide

THF Tetrahydrofuran

DIEA N,N-Diisopropylethylamine

DTT Dithiothreitol

EtOH Ethanol

MeOH Methanol

For synthesis of compounds 1-71, All reagents were purchased from commercial sources and used without further puriﬁcation.

Unless otherwise stated, the commercial source all commonly available reagents was Sigma-Aldrich Corp. (St. Louis, MO, USA),

VWR (Radnor, PA, USA), or Matrix Scientiﬁc (Elgin, SC, USA).

Scheme 1. Preparation of 6a – 6n.

General Procedure A (Scheme 1), amino-1H-pyrazoles (6a – 6n)

Ethyl-2-cyano-3-ethoxyacrylate (1.50 g, 8.87 mmol, 1.0 equiv.) (Sigma-Aldrich, Cat# E28005) was combined with N,N-diisopropyle-

thylamine (1.7 mL, 9.8 mmol, 1.1 equiv.) and the appropriate hydrazine (9.8 mmol, 1.1 equiv.) in ethanol (10 mL). The mixture was

heated to 80^ꢁC for 12 hours or until starting hydrazine was consumed. Silica gel chromatography or crystallization yielded 6a – 6n.

Ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (6a)

General procedure A was followed using phenylhydrazine (Sigma-Aldrich, Cat# P26252). The mixture was condensed to dryness

and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (20-40% ethyl acetate in hexane) yielded

6a (96% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₂H₁₃N₃O₂232.0; found 232.0.

Ethyl 5-amino-1-(2-chlorophenyl)-1H-pyrazole-4-carboxylate (6b)

General procedure A was followed using 2-chlorophenylhydrazine hydrochloride (Sigma-Aldrich, Cat# 109509). The mixture

was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (20-40% ethyl

acetate in hexane) yielded 6b (99% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₂H₁₂ClN₃O₂266.1; found 266.0.

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Ethyl 5-amino-1-(3-chlorophenyl)-1H-pyrazole-4-carboxylate (6c)

General procedure A was followed using 3-chlorophenylhydrazine hydrochloride (Matric Scientiﬁc, Cat# 075249). The mixture was

condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (0-50% ethyl acetate

in hexane) yielded 6c (86% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₂H₁₂ClN₃O₂266.1; found 266.0.

Ethyl 5-amino-1-(4-chlorophenyl)-1H-pyrazole-4-carboxylate (6d)

General procedure A was followed using 4-chlorophenylhydrazine hydrochloride (Sigma-Aldrich, Cat# C65807). The mixture was

condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (0-20% ethyl acetate

in dichloromethane) yielded 6d (95% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₂H₁₂ClN₃O₂266.1; found 266.0.

Ethyl 5-amino-1-isopropyl-1H-pyrazole-4-carboxylate (6e)

General procedure A was followed using isopropylhydrazine hydrochloride (Matrix Scientiﬁc, Elgin, SC, USA, Cat# 007266).

The mixture was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution

(20-40% ethyl acetate in hexane) yielded 6e (95% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₉H₁₅N₃O₂198.1; found 198.0.

Ethyl 5-amino-1-(o-tolyl)-1H-pyrazole-4-carboxylate (6f)

General procedure A was followed using o-tolylhydrazine hydrochloride (Alfa Aesar, Cat# A15767). The mixture was condensed to

dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (20-30% ethyl acetate in hexane)

yielded 6f (99% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₅N₃O₂246.1; found 246.1.

Ethyl 5-amino-1-(m-tolyl)-1H-pyrazole-4-carboxylate (6g)

General procedure A was followed using m-tolylhydrazine hydrochloride (Acros, Cat# 139150010). The mixture was condensed to

dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution (20-30% ethyl acetate in hexane)

yielded 6g (86% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₅N₃O₂246.1; found 246.1.

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Ethyl 5-amino-1-(3-methoxyphenyl)-1H-pyrazole-4-carboxylate (6h)

General procedure A was followed using 3-methoxyphenylhydrazine hydrochloride (Matrix Scientiﬁc, Elgin, SC, USA, Cat#

021395). The mixture was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient

elution (20-30% ethyl acetate in hexane) yielded 6h (53% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₅N₃O₃262.1;

found 262.1.

Ethyl 5-amino-1-(4-methoxyphenyl)-1H-pyrazole-4-carboxylate (6i)

General procedure A was followed using 4-methoxyphenylhydrazine hydrochloride (Matrix Scientiﬁc, Elgin, SC, USA, Cat#

021131). The mixture was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient

elution (20-40% ethyl acetate in hexane) yielded 6i (86% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₅N₃O₃262.1;

found 262.1.

Ethyl 5-amino-1-(3-cyanophenyl)-1H-pyrazole-4-carboxylate (6j)

General procedure A was followed using 3-cyanophenylhydrazine hydrochloride (Ark Pharm, Inc., AK-39869). The mixture

was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient elution

(0-40% ethyl acetate in hexane) yielded 6j (71% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₂N₄O₂257.1; found

257.1.

Ethyl 5-amino-1-(4-cyanophenyl)-1H-pyrazole-4-carboxylate (6k)

General procedure A was followed using 4-cyanophenylhydrazine hydrochloride (Sigma-Aldrich, Cat# 453471). Addition of

water (5 mL) to the reaction mixture resulted in precipitation of an off white solid, which was isolated by ﬁltration and

washed with ethanol and water to yield 6k (94% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₂N₄O₂257.1; found

257.1.

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Ethyl 5-amino-1-(3-ﬂuorophenyl)-1H-pyrazole-4-carboxylate (6l)

General procedure A was followed using 3-ﬂuorophenylhydrazine hydrochloride (Matrix Scientiﬁc, Elgin, SC, USA, Cat# 003925).

The mixture was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient

elution (10-20% ethyl acetate in hexane) yielded 6l (88% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₂H₁₂FN₃O₂250.1;

found 250.1.

Ethyl 5-amino-1-(3-(triﬂuoromethyl)phenyl)-1H-pyrazole-4-carboxylate (6m)

General procedure A was followed using 3-(triﬂuoromethyl)phenylhydrazine hydrochloride (Matrix Scientiﬁc, Elgin, SC, USA, Cat#

005466). The mixture was condensed to dryness and dissolved in dichloromethane (5 mL). Silica gel chromatography with gradient

elution (10-20% ethyl acetate in hexane) yielded 6m (84% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₂F₃N₃O₂300.1;

found 300.1.

4-(5-amino-4-(ethoxycarbonyl)-1H-pyrazol-1-yl)benzoic acid (6n)

General procedure A was followed using 4-hydrazinobenzoic acid hydrochloride (Alfa Aesar, Ward Hill, MA, USA, Cat# B20326). The

mixture was condensed to dryness and dissolved in dichloromethane with 10% methanol (5 mL). Silica gel chromatography with

gradient elution (0-16% methanol in dichloromethane) yielded 6n (99% yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for

C₁₃H₁₃N₃O₄276.1; found 276.1.

Scheme 2. Preparation of 7a – 7n.

General Procedure B (Scheme 2), 6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7a – 7n)

Amino-1H-pyrazoles (6a – 6n) (3 – 14 mmol, 1.0 equiv.) were combined with benzoyl isothiocyanate (1.2 equiv.) in tetra-

hydrofuran (50 mL). The heated to reﬂux under Ar(g) until starting material was consumed (6 – 18 hours). The resulting thiourea

was added dropwise to a reﬂuxing solution of ethanol (50 mL) and sodium ethoxide (21 wt. % in ethanol, 10 mL). The mixture

was stirred for 30 min. at 90^ꢁC and condensed to dryness. Product was precipitated by the addition of water (50 mL).

After stirring overnight, the off white solid was isolated by ﬁltration and washed with water, then 50% ether in heptane to

give 7a – 7n.

e11 Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018

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6-mercapto-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7a)

General procedure B was followed using 6a (1.0 g, 4.3 mmol) to produce 7a (0.91 g, 86 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₁H₈N₄OS 245.1; found 245.0.

1-(2-chlorophenyl)-6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7b)

General procedure B was followed using 6b (1.2 g, 4.5 mmol) to produce 7b (0.92 g, 73 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₁H₇ClN₄OS 279.0; found 279.0.

1-(3-chlorophenyl)-6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7c)

General procedure B was followed using 6c (3.8 g, 14.3 mmol) to produce 7b (3.7 g, 92 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₁H₇ClN₄OS 279.0; found 279.0.

1-(4-chlorophenyl)-6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7d)

General procedure B was followed using 6d (0.86 g, 3.2 mmol) to produce 7b (0.65 g, 72 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₁H₇ClN₄OS 279.0; found 279.0.

1-isopropyl-6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7e)

General procedure B was followed using 6e (1.6 g, 8.4 mmol) to produce 7e (0.88 g, 50 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₈H₁₀N₄OS 211.1; found 211.0.

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6-mercapto-1-(o-tolyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7f)

General procedure B was followed using 6f (2.2 g, 9.0 mmol) to produce 7e (1.95 g, 84 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₂H₁₀N₄OS 259.1; found 259.1.

6-mercapto-1-(m-tolyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7g)

General procedure B was followed using 6g (1.8 g, 7.2 mmol) to produce 7g (1.63 g, 86 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₂H₁₀N₄OS 259.1; found 259.1.

6-mercapto-1-(3-methoxyphenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7h)

General procedure B was followed using 6h (1.2 g, 4.7 mmol) to produce 7h (0.83 g, 65 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₂H₁₀N₄O₂S 275.1; found 275.1.

6-mercapto-1-(4-methoxyphenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7i)

General procedure B was followed using 6i (2.0 g, 7.7 mmol) to produce 7i (2.01 g, 96 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd

for C₁₂H₁₀N₄O₂S 275.1; found 275.1.

Synthesis of 3-(6-mercapto-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile (7j)

General procedure B was followed using 6j (2.2 g, 8.4 mmol) to produce 7j (1.1 g, 50 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd

for C₁₂H₇N₅OS 270.0; found 270.0.

e13 Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018

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4-(6-mercapto-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile (7k)

General procedure B was followed using 6k (2.1 g, 8.3 mmol) to produce 7k (2.1 g, 96 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd

for C₁₂H₇N₅OS 270.0; found 270.0.

1-(3-ﬂuorophenyl)-6-mercapto-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7l)

General procedure B was followed using 6l (1.0 g, 4.2 mmol) to produce 7l (0.80 g, 73 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd

for C₁₁H₇FN₄OS 263.0; found 263.0.

6-mercapto-1-(3-(triﬂuoromethyl)phenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7m)

General procedure B was followed using 6m (0.60 g, 2.0 mmol) to produce 7m (0.53 g, 85 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₂H₇F₃N₄OS 313.0; found 313.1.

4-(6-mercapto-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzoic acid (7n)

General procedure B was followed using 6n (0.14 g, 0.52 mmol) to produce 7n (0.064 g, 42 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺

calcd for C₁₂H₈N₄O₃S 289.0; found 289.0.

Scheme 3. Preparation of 2, 8-44.

2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)acetonitrile, HS56 (2)

To a solution of 7c (1.0 g, 3.6 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (10 mL) with N,N-diisopropylethylamine (1.25 mL,

7.2 mmol, 2.0 equiv.) under Ar(g) was added chloroacetonitrile (0.227 mL, 3.6 mmol, 1.0 equiv.) dropwise over 2 minutes. After stirring

for 30 minutes the reaction was condensed to a tan oil and stirred under 50% ethyl acetate in heptane (50 mL) for 5 hours.

The resulting off white precipitate was collected by ﬁltration and washed 50% ethyl acetate in heptane followed by water to give

HS56 (2) (1.05 g, 83 % yield). ¹H NMR (500 MHz, dmso-d₆) d (ppm): 13.07 (s, 1H), 8.33 (s, 1H), 8.23 (s, 1H), 8.16 (d, J = 7.1 Hz,

Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018 e14

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1H), 7.57 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 4.36 (s, 2H); ¹³C NMR (500 MHz, dmso-d₆) d (ppm): 158.9, 157.1, 151.2, 139.2,

136.7, 133.7, 130.9, 126.8, 120.6, 119.6, 117.3, 105.2, 16.4.

1-(3-chlorophenyl)-6-((2-hydroxyethyl)thio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, HS43 (8)

Compound 8 was prepared as previously described (Carlson et al., 2013). To a solution of 7c (0.10 g, 0.36 mmol, 1.0 equiv.)

in anhydrous dimethylformamide (1.0 mL) with N,N-diisopropylethylamine (0.188 mL, 1.1 mmol, 3.0 equiv.) was added 2-bromoe-

thanol (0.028 mL, 0.39 mmol, 1.1 equiv.). The reaction was stirred for 12 hours and formic acid (0.30 mL) was added.

The acidiﬁed mixture was puriﬁed by preparative HPLC (40 - 100 % methanol in water with 0.2 % formic acid modiﬁcation

over 18 minutes) to give HS43 (8) (0.067 g, 58 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₃H₁₁ClN₄O₂S 323.0;

found 323.1.

1-(3-chlorophenyl)-6-(isobutylthio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, HS44 (9)

To a solution of 7c (0.040 g, 0.14 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine (0.075 mL,

0.43 mmol, 3.0 equiv.) was added 1-bromo-2-methylpropane (0.062 mL, 0.57 mmol, 4.0 equiv.). The reaction was heated to 30^ꢁC for

4 hours, condensed to remove solvent, and dissolved in DMSO. Preparative HPLC (5 - 100 % methanol in water with 0.2 % formic

acid modiﬁcation over 20 minutes) provided HS44 (9) (0.010 g, 21 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₅H₁₅ClN₄OS

335.1; found 335.1.

Ethyl 4-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)butanoate, HS49 (10)

To a solution of 7c (0.040 g, 0.14 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine (0.075 mL,

0.43 mmol, 3.0 equiv.) was added ethyl 4- bromobutyrate (0.083 mL, 0.57 mmol, 4.0 equiv.). The reaction was heated to 30^ꢁC for 4

hours, condensed to remove solvent, and dissolved in DMSO. Preparative HPLC (5 - 100 % methanol in water with 0.2 % formic acid

modiﬁcation over 20 minutes) provided HS49 (10) (0.016 g, 28 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₇H₁₇ClN₄O₃S

393.1; found 393.2.

2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)-2-methylpropanoic acid, HS50 (11)

To a solution of 7c (0.040 g, 0.14 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine (0.075 mL,

0.43 mmol, 3.0 equiv.) was added 2-bromo-2-methylpropionic acid (0.096 g, 0.57 mmol, 4.0 equiv.). The reaction was heated to 30^ꢁC

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for 4 hours, condensed to remove solvent, and dissolved in DMSO. Preparative HPLC (5 - 100 % methanol in water with 0.2 % formic

acid modiﬁcation over 20 minutes) provided HS50 (11) (0.032 g, 61 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for

C₁₅H₁₃ClN₄O₃S 365.0; found 365.2.

Ethyl 4-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)-3-oxobutanoate, HS51 (12)

To a solution of 7c (0.040 g, 0.14 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine (0.075 mL,

0.43 mmol, 3.0 equiv.) was added ethyl 4-chloroacetoacetate (0.079 mL, 0.57 mmol, 4.0 equiv.). The reaction was heated to 30^ꢁC for

4 hours, condensed to remove solvent, and dissolved in DMSO. Preparative HPLC (5 - 100 % methanol in water with 0.2 % formic

acid modiﬁcation over 20 minutes) provided HS51 (12) (0.031 g, 53 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for

C₁₇H₁₅ClN₄O₄S 407.1; found 407.2.

1-(3-chlorophenyl)-6-((3-hydroxypropyl)thio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, HS53 (13)

To a solution of 7c (0.050 g, 0.18 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine

(0.094 mL, 0.54 mmol, 3.0 equiv.) was added 3-bromo-1-propanol (0.017 mL, 0.20 mmol, 1.1 equiv.). The reaction was heated

to 30^ꢁC for 12 hours and then partitioned between water (2 mL) and ethyl acetate (2 mL). The resulting precipitate was isolated

by ﬁltration to give HS53 (13) (0.044 g, 73 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₄H₁₃ClN₄O₂S 337.1;

found 337.1.

1-(3-chlorophenyl)-6-((2-oxo-2-phenylethyl)thio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, HS54 (14)

To a solution of 7c (0.050 g, 0.18 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine

(0.094 mL, 0.54 mmol, 3.0 equiv.) was added 2-bromoacetophenone (0.039 g, 0.20 mmol, 1.1 equiv.). The reaction was heated

to 30^ꢁC for 12 hours and then partitioned between water (2 mL) and ethyl acetate (2 mL). The resulting precipitate was isolated

by ﬁltration to give HS54 (14) (0.046 g, 65 % yield). LCMS (ESI Ion Trap) m/z: [M+H]⁺calcd for C₁₉H₁₃ClN₄O₂S 397.1;

found 397.2.

1-(3-chlorophenyl)-6-(((6-chloropyridin-3-yl)methyl)thio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, HS58 (15)

To a solution of 7c (0.050 g, 0.18 mmol, 1.0 equiv.) in anhydrous dichloromethane (1.0 mL) with N,N-diisopropylethylamine

(0.094 mL, 0.54 mmol, 3.0 equiv.) was added 2-chloro-5-(chloromethyl)pyridine (0.032 g, 0.20 mmol, 1.1 equiv.). The reaction

Cell Chemical Biology 25, 1–13.e1–e32, September 20, 2018 e16

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Targeting Pim Kinases and DAPK3 to Control Hypertension

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