Target-Directed Self-Assembly of Homodimeric Drugs Against β-Tryptase

Article abstract of DOI:10.1021/acsmedchemlett.8b00204

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC₅₀ (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

Full text of DOI:10.1021/acsmedchemlett.8b00204

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Innovations
Target-Directed Self-Assembly of Homodimeric Drugs Against #-Tryptase
Sarah F. Giardina, Douglas S Werner, Maneesh R Pingle, Kenneth
W Foreman, Donald E Bergstrom, Lee D. Arnold, and Francis Barany
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00204 • Publication Date (Web): 05 Jul 2018
Downloaded from http://pubs.acs.org on July 6, 2018
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ACS Medicinal Chemistry Letters
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TargetꢀDirected SelfꢀAssembly of Homodimeric Drugs Against βꢀ
Tryptase
Sarah F. Giardina†,*, Douglas S. Werner‡,§, Maneesh Pingle†,‡,§, Kenneth W. Foreman‡,║, Donald E.
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Bergstrom⊥, Lee D. Arnold‡,∇ and Francis Barany†
† Department of Microbiology and Immunology, Weill Cornell Medicine, 1300 York Ave, Box 62. New York, NY, 10065
‡ Coferon, Inc., 25 Health Sciences Drive, Mailbox 123, Stony Brook, NY, 11790.
§ Present Address: BlinkBio, Inc., The Scripps Research Institute, 130 Scripps Way #2B2, Jupiter, FL, 33458.
⊥ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall, West Lafayette,
IN, 47907.
║ Present Address: Ars Exemplar, LLC, Syosset, NY.
∇Present Address: Fount Therapeutics, LLC, Wilmington, New Castle, DE, 19801.
KEYWORDS coferon, small molecule, tryptase inhibitor, drug discovery, bivalent, homodimer
ABSTRACT: Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Huꢀ
man tryptase is donutꢀshaped tetramer with the active sites facing inwards forming a central pore. Bivalent ligands spanning two
active sites potently inhibit this configuration, but these large compounds have poor drugꢀlike properties. To overcome some of
these challenges, we developed selfꢀassembling molecules, called coferons, which deliver a larger compound in two parts. Using a
pharmacophoric core and reversibly binding linkers to span two active sites we have successfully produced three novel homodimerꢀ
ic tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements
in IC50 (0.19 µM ± 0.08) over controls (5.50 µM ± 0.09), and demonstrate good activity in mast cell lines. These studies provide
validation for this innovative technology that is especially wellꢀsuited for the delivery of dimeric drugs to modulate intracellular
macromolecular targets.
One of the most formidable and fundamental challenges of
small molecule drug design is to effectively and specifically
target specific intracellular proteins 1ꢀ2. Often small molecule
ligands cannot achieve sufficiently high affinities to disrupt
macromolecular interactions through localized binding sites
and extending their “reach” to exploit additional pharmacoꢀ
phoric contacts results in larger multivalent molecules with
subꢀoptimal drug properties 3. To circumvent these limitations,
we have developed coferons, reversibly selfꢀassembling drug
molecules that may be administered as a dimer, which dissociꢀ
ates into monomers in vivo for absorption, distribution, and
permeation into tissues and cells. Upon binding to their macꢀ
romolecular target, coferon monomers covalently reꢀassociate
to form a larger, more specific, and higherꢀaffinity dimeric
assembly to effectively modulate interactions or activity. Each
coferon monomer is comprised of an appropriate ‘pharmacoꢀ
phore’, with an affinity for a binding site on the target, conꢀ
nected to a small linker moiety capable of reversible, covalent
reaction with a ‘partner’ coferon binding at a proximal site on
In conjoining two weaker pharmacophores into a higher afꢀ
finity assembly, the coferon concept incorporates key attribꢀ
5ꢀ7
utes of multivalency
and fragmentꢀbased drug discovery
(FBDD) 8ꢀ9, and in using the biomolecular target as a template,
also emulates proteinꢀdirected dynamic combinatorial chemisꢀ
try (DCC) 10ꢀ13. Our efforts build upon the early precedents of
weakly active drug fragments assembling covalently in situ (in
cells) to form more active entities 14, and target directed selfꢀ
assembly of higher affinity ligands and inhibitors 12, 15ꢀ17. Our
approach is uniquely differentiated by the use of reversibly
covalent, bioorthogonal linker moieties to enable drug delivery
and subsequent in situ dimerization of pharmacophoric comꢀ
ponents upon the protein target inside cells.
Although a large number of potential protein interfaces
could be considered for the validation of this technology, mast
cell βꢀtryptase offered a number of advantages. Tryptase is a
tetrameric trypsinꢀfamily protease that is the most abundant
protein in human mast cells 18, and its release from granules
has been linked to the pathology and progression in allergic
and inflammatory disease 19ꢀ24. Bivalent inhibitors spanning
approximately 30 Å that engage pharmacophore binding sites
of two adjacent subunits have demonstrated more than 1,000ꢀ
fold increase in selectivity and affinity, and have shown effiꢀ
cacy in preclinical models and clinical studies 25ꢀ26. However,
the large size, doubleꢀcharge and physicochemical properties
4
the target . Thus, the macromolecular biological target serves
as a template for the assembly of the higher affinity coferon
dimer. The delivery of a larger dimeric drug molecule as two
low molecular weight monomeric coferons affords greater
flexibility in optimizing drugꢀlike absorption, distribution,
metabolism, and excretion (ADME) properties.
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of these molecules produced poor drugꢀlike performance and
precluded good bioavailability 25, 27ꢀ29
was 10.9 nM at 10 pM tryptase while the potency of the monꢀ
1
2
3
4
5
6
7
8
.
omeric diols was affected minimally (Table 1). The impressive
potency enhancement of 3a was also reflected in tryptase celꢀ
lular degranulation assays (IC50 = 52 nM) and cell lysates from
cultured HMC1 human mast cells (IC50 = 113 nM; Table 2).
Xꢀray coꢀcrystallographic studies supported the proposed hoꢀ
modimeric mechanism of tryptase inhibition for 1a, 2a, and 3a,
with contiguous electron density bridging two proximal pharꢀ
macophore binding sites (Table S1). For the most potent anaꢀ
log, 3a, the observed electron density could be accounted for
by a single spiroketal diastereomer bound in both directions
(Fig. 2b, Fig S1a), but for 1a, and 2a contributions from multiꢀ
ple diastereomeric spiroketal assemblies were evident (Fig.
2c). This illustrates how the biomolecular target may promote
the formation of one or more homodimeric assemblies with
similar energies. Protein Data Bank (PDB): coordinates and
structure factors for the coꢀcrystal structures of the tryptase
complexes with 2a and 3a have been deposited with accession
codes 4MPU and 4MPV, respectively.
Herein we demonstrate targetꢀdirected selfꢀassembling of
homodimeric inhibitors of human βꢀtryptase and achieve
greatly enhanced affinity. Our preliminary evidence using αꢀ
hydroxyketoꢀbased homodimeric linkers supports the concept,
realizing the advantage of bivalent inhibitors and multivalency
7 without compromising cellular permeability.
Simulations suggested that micromolar affinity pharmacoꢀ
phores could combine to achieve subꢀnanomolar inhibition,
corresponding to potency enhancements in excess of several
thousandꢀfold if certain reasonable criteria are met. We refer
to this potency enhancement as the “coferon effect,” calculatꢀ
ed as the ratio of the IC50 of the control monomer and the apꢀ
parent coferon IC50. Improvements in apparent IC50s are exꢀ
pected to be proportional to the root of the coferon dimerizaꢀ
tion, and directly proportional to enhancements in monomer
affinities until the tightꢀbinding limit is approached. Interestꢀ
ingly, as expected for tightꢀbinding inhibitors, the apparent
IC50 for the coferons is predicted to decrease (with a correꢀ
sponding increase in coferon effect) with decreasing target
concentration.
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The high stability of tryptase’s proteolytic activity at room
temperature enabled reversibility studies of compounds to be
conducted over an extended period of time. After the removal
of excess unbound inhibitor from tryptase by a gel filtration
spinꢀcolumn we monitored the recovery of enzymic activity.
Monomeric inhibitors were readily dissociated under these
conditions to immediately restore full tryptase activity, while
less than 25% of activity was recovered after 9 days with
compound 1a (Fig S1c).
To facilitate the design, exploration, and development of
coferon linker chemistries to enable the selfꢀassembly techꢀ
nology for inhibition of tetrameric βꢀtryptase, we selected the
[3ꢀ(1ꢀacylpiperidinꢀ4ꢀyl)phenyl]methanamine moiety (Fig. 1a)
as the pharmacophoric core. Relatively simple variants of this
pharmacophore produce monovalent tryptase inhibitors with
potencies in the 10ꢀ6 to 10ꢀ8 M range with the N1ꢀacyl moieties
Through the use of computer simulations based upon basic
equilibrium models and relatively simple homodimeric linker
chemistries, we have established the foundation for the delivꢀ
ery of bivalent drugs, based on the targetꢀdirected and reversiꢀ
ble covalent assembly of monomeric coferons. Our approach
allows significant flexibility for optimizing monomers for
improved permeability, metabolic stability, and pharmacokiꢀ
netics while delivering the superior potency of the larger diꢀ
meric payload.
26,
bound proximally in adjacent catalytic subunits
30. Imꢀ
portantly, achieving suitable dimer dissociation constants (i.e.
KDim < 10ꢀ2 M) with small linker moieties to produce signifiꢀ
cant coferon effects precluded the use of weak noncovalent
bonding interactions 31 and compelled us to explore reversibly
covalent αꢀhydroxyketo linker chemistries (Fig. 1b).
Our homodimeric coferon designs explored simple αꢀ
hydroxyketo (αꢀHK) moieties as linkers 32. The idea was supꢀ
ported by the knowledge that dihydroxyacetone exists as a
dimer in concentrated solutions and in the solid state 33, and
reports that αꢀHK mineroloꢀ and glucoꢀcorticoid progestagens
form dimers in aqueous solutions 34. Of the regioisomeric αꢀ
HK dimer assemblies that could be produced, several diastereꢀ
omeric 5ꢀmembered spiroketal (1,3ꢀdioxolanꢀ4ꢀol) linkages
(Fig. 1) are predicted to be thermodynamically favored over
cyclic 6ꢀmembered bisꢀhemiketals (i.e. 1,4ꢀdioxaneꢀ2,5ꢀdiol).
Our initial design based upon αꢀhydroxyacetonyl ether (R, R’=
H in Fig. 1b); IC50 = 49 nM at 1 nM tryptase) displayed a marꢀ
ginal 5ꢀfold improvement in activity compared to its nonꢀ
dimerizable diol analog, however the linker moiety proved to
be unstable in aqueous buffers and plasma due to tautomerizaꢀ
tion to the enediol, and hydrolytic fragmentation 35. This issue
was circumvented through the geminal dialkyl substitution of
the αꢀhydroxymethyl moiety to block the tautomerization,
discourage oxidation, and inhibit metabolic conjugation.
The current study uses a novel chemistry platform to estabꢀ
lish selfꢀassembling homodimeric inhibitors of tryptase. Our
efforts have demonstrated the attributes of synthetic ease of
introduction, effectiveness, specificity, kinetic facility, biologꢀ
ical inertness, and physiologic compatibility of the linkers,
while minimally perturbing the affinity and drugꢀlike behavior
of the pharmacophoric moiety to which it is appended. Thus,
we anticipate that this technology will provide a versatile,
broadly applicable linker, deployable in diverse structural arꢀ
rangements for drug discovery and delivery of bivalent moleꢀ
cules with improved cellular permeability. Our coferon dimers
are sufficiently stable to be chromatographed, crystallized, and
stored without decomposition, making them suitable as active
pharmaceutical constituents. Thus, we anticipate that our
coferon platform will provide a versatile, and broadly applicaꢀ
ble coferon linker for drug discovery and delivery efforts, and
amenable to tuning and engineering properties through entropꢀ
ic, electronic and steric effects 4.
The αꢀhydroxyacetonyl 1a (Fig.1b, R, R’ = ꢀ(CH2)3ꢀ), and
the αꢀhydroxypyruvylamido 2a (Fig.1b, R, R’ = ꢀ(CH2)3ꢀ) deꢀ
rivatives exhibited improved stability under aqueous condiꢀ
tions, and significant improvements in IC50 in vitro of over
200ꢀfold of their nonꢀdimerizable racemic vicinal diol analogs
(1b, 2b, 3b, respectively) (Fig.2a, Fig S1b). As predicted from
simulations the in vitro IC50s of these coferons improved with
decreasing tryptase concentrations, such that the IC50 for 3a
Experimental Procedures
For details regarding the synthesis of the compounds and exꢀ
perimental procedures see supplemental methods.
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Figure 1. The designs for targetꢀdirected selfꢀassembling dimeric
drugs.
One monomer binds each catalytically active subunit of tryptase,
such that each homodimeric coferon inhibits an adjacent pair of
tryptase subunits. (B) Examples of homodimerizing connector
plus linker moieties explored in the current manuscript.
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Figure 1. (A) [3ꢀ(1ꢀAcylpiperidinꢀ4ꢀyl)phenyl]methanamine was
employed as the pharmacophoric core for the development of
reversible homodimeric inhibitors of tetrameric human βꢀtryptase.
IC50 [M] ± SEM
IC50 Improvement over
control
(Coferon Effect)
1a/1b 2a/2b 3a/3b
aꢀHK #
2a
Diol Control #
2b
1a
3a
1b
3b
2.3 x 10ꢀ8 ± 1.0 x 10ꢀ8 ± 2.7 x 10ꢀ9 ± 6.6 x 10ꢀ6 ± 6.4 x 10ꢀ7 ± 4.9 x 10ꢀ7 ±
10 pM
100 pM
1 nM
61.7
39.1
43.1
177.8
208.3
13.1
282.8
19.6
8.5
0.06
1.0 x 10ꢀ7 ± 4.2 x 10ꢀ8 ± 2.1 x 10ꢀ8 ± 2.0 x 10ꢀ6 ± 1.7 x 10ꢀ6 ± 4.3 x 10ꢀ6 ±
0.15 0.13 0.05 0.15 0.09 0.14
3.7 x 10ꢀ7 ± 1.9 x 10ꢀ7 ± 3.7 x 10ꢀ7 ± 3.1 x 10ꢀ6 ± 8.1 x 10ꢀ6 ± 4.9 x 10ꢀ6 ±
0.04 0.04 0.05 0.04 0.05 0.06
0.07
0.11
0.26
0.14
0.15
Table 1 IC50s for the αꢀHydroxyketo homodimers.
Table 1. αꢀHydroxyketo coferons demonstrate a concentrationꢀdependent increase in potency over their vicinal diol analogs in assays with
purified enzyme. IC50s for the homodimers decreased with decreasing target concentration and corresponded to an increase in fold imꢀ
provements. Fold difference was determined from the monomeric diol analogs. Intensity of red boxes indicates the degree of fold differꢀ
ence. IC50s were determined from nonlinear regression, with no constraints on Hill slope using Graphpad prism.
Figure 2. Tryptase promotes the assembly of αꢀhydroxyketoꢀbased coferon dimers resulting in improved potency.
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Figure 2. (A) αꢀHydroxyketo coferons 1a, 2a, and 3a display significant in vitro and cellular potency improvements (ratios) over their nonꢀ
dimerizable racemic vicinal diol analogs (1b, 2b and 3b, respectively). (B) The coꢀcrystal structure of 3a at 2.3 Å resolution with human β–
tryptase indicates that pharmacophore binding sites in adjacent subunits are bridged by a dimeric spiroketal assembly. The (2R,4S)ꢀ4ꢀ
hydroxyꢀ2ꢀ(1ꢀhydroxyꢀ1ꢀmethylꢀethyl)ꢀ1,3ꢀdioxolane diastereomer in leftꢀtoꢀright, and rightꢀtoꢀleft configuration (depicted displayed on
protein surface colored by electrostatic character) was best suited in fitting the bridging density. (C) Contiguous electron density was also
observed in the coꢀcrystal structure of 2a (1.65 Å resolution) consistent with occupancy by dimeric spiroketal assemblies. While, an R,Sꢀ
diastereomer, analogous to that of 3a, contributes to the observed 1σ density (depicted), no single spiroketal configuration could account
for all aspects (Table S1).
Table 2 αꢀHydroxyketo coferons demonstrate good potency in degranulation and whole lysate assays in HMC1 cells.
IC50 in HMC1 Cellular Degranulation [M]
Diol Conꢀ
IC50 in HMC1 Cellular Lysates [M]
Diol Conꢀ
aꢀHK #
IC50 [M]
SEM
SEM
IC50 [M]
SEM
SEM
trol
trol
4.33 x 10ꢀ7
4.39 x 10ꢀ7
5.20 x 10ꢀ8
0.4
1.08 x 10ꢀ5
1.37 x 10ꢀ4
3.15 x 10ꢀ5
0.07
0.41
0.1
4.54 x 10ꢀ7
3.14 x 10ꢀ7
1.13 x 10ꢀ7
0.12
0.06
0.05
8.40 x 10ꢀ6
2.18 x 10ꢀ5
1.33 x 10ꢀ5
0.07
0.11
0.1
1a
2a
3a
0.19
0.43
Table 2. Cells treated with inhibitors (10 nMꢀ100 µM; 2h) were degranulated in the presence of 1 µM of A23187 in PBS. After 1h the
supernatant was assayed for tryptase activity. Alternatively, IC50s were determined in cell lysates. IC50s and S.E.M. were determined from
nonlinear regression, with no constraints on Hill slope using Graphpad prism.
Department of Microbiology and Immunology
1300 York Ave, Box 62
New York, NY, 10021
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Phone: 2127466507
Facsimile: 2127467983
Email: sfg2001@med.cornell.edu
For details regarding the synthesis of the compounds see suppleꢀ
mental methods (PDF).
Author Contributions
AUTHOR INFORMATION
Corresponding Author
Study concept and design: SFG and LDA. MP, FB and DEB deꢀ
veloped the Coferon concept and LDA, DEB and MP developed
the linker chemistries. DSW performed and oversaw syntheses.
Acquisition of data: SFG. Analysis and interpretation of data:
*Sarah Giardina
Weill Cornell Medicine
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ACS Medicinal Chemistry Letters
13.
Bhat, V. T.; Caniard, A. M.; Luksch, T.; Brenk, R.;
SFG, LDA, DWS, KWF. SFG, LDA and DSW wrote the manuꢀ
script. All authors have given approval to the final version of the
manuscript.
Campopiano, D. J.; Greaney, M. F., Nucleophilic catalysis of acylhyꢀ
drazone equilibration for proteinꢀdirected dynamic covalent chemisꢀ
try. Nat Chem 2010, 2, 490ꢀ7.
1
2
3
Funding Sources
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Rideout, D., Selfꢀassembling cytotoxins. Science 1986,
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233, 561ꢀ3.
This work was supported in part by the Center for Biotechnology,
an Empire State Development Division of Science, Technology &
Innovation Designated Center for Advanced Technology. F.B.,
S.F.G., M.P., D.E.B. were supported by a grant from Coferon Inc.
15.
Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carꢀ
lier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B., Click chemistry
in situ: acetylcholinesterase as a reaction vessel for the selective asꢀ
sembly of a femtomolar inhibitor from an array of building blocks.
Angew Chem Int Ed 2002, 41, 1053ꢀ7.
ACKNOWLEDGMENT
16.
Katz, B. A.; FinerꢀMoore, J.; Mortezaei, R.; Rich, D. H.;
The authors thank D.A. Beard (Medical College of Wisconsin)
and R.C. Jackson (Pharmacometrics, UK) for kinetic and compuꢀ
tational data analyses and discussions; A. White and R. Sato of
Xtal BioStructures, Inc. for production of cocrystals and Xꢀray
structure elucidation; Sai LifeScience for synthesis, equilibria
analyses, and pharmacokinetic studies.
Stroud, R. M., Episelection: Novel Ki ~ Nanomolar Inhibitors of
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ABBREVIATIONS
αꢀHK, αꢀhydroxyketo; FBDD, fragmentꢀbased drug discovery;
AMC 7ꢀaminoꢀ4ꢀmethylcoumarin.
19.
Cairns, J. A., Inhibitors of mast cell tryptase beta as theraꢀ
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Gosman, M. M.; Postma, D. S.; Vonk, J. M.; Rutgers, B.;
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Figure 1. (A) [3ꢀ(1ꢀAcylpiperidinꢀ4ꢀyl)phenyl]methanamine was employed as the pharmacophoric core for
the development of reversible homodimeric inhibitors of tetrameric human βꢀtryptase. One monomer binds
each catalytically active subunit of tryptase, such that each homodimeric coferon inhibits an adjacent pair of
tryptase subunits. (B) Examples of homodimerizing connector plus linker moieties explored in the current
manuscript.
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Figure 2. (A) αꢀHydroxyketo coferons 1a, 2a, and 3a display significant in vitro and cellular potency
improvements (ratios) over their nonꢀdimerizable racemic vicinal diol analogs (1b, 2b and 3b, respectively).
(B) The coꢀcrystal structure of 3a at 2.3 Å resolution with human β–tryptase indicates that pharmacophore
binding sites in adjacent subunits are bridged by a dimeric spiroketal assembly. The (2R,4S)ꢀ4ꢀhydroxyꢀ2ꢀ
(1ꢀhydroxyꢀ1ꢀmethylꢀethyl)ꢀ1,3ꢀdioxolane diastereomer in leftꢀtoꢀright, and rightꢀtoꢀleft configuration
(depicted displayed on protein surface colored by electrostatic character) was best suited in fitting the
bridging density. (C) Contiguous electron density was also observed in the coꢀcrystal structure of 2a (1.65 Å
resolution) consistent with occupancy by dimeric spiroketal assemblies. While, an R,Sꢀdiastereomer,
analogous to that of 3a, contributes to the observed 1σ density (depicted), no single spiroketal configuration
could account for all aspects (Table S1).
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Target-Directed Self-Assembly of Homodimeric Drugs Against
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β-Tryptase
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Sarah F. Giardina†,*, Douglas S. Werner‡,§, Maneesh Pingle†,‡,§, Kenneth W. Foreman‡,║, Donald
E. Bergstrom⊥, Lee D. Arnold‡,∇ and Francis Barany†
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