Exploring Ligand-Directed N-Acyl- N-alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development

Acylation Chemistry for Potential Targeted Degrader Development

Letter

Exploring Ligand-Directed N‑Acyl‑N‑alkylsulfonamide-Based

Mingxing Teng, Jie Jiang, Scott B. Ficarro, Hyuk-Soo Seo, Jae Hyun Bae, Katherine A. Donovan,

Eric S. Fischer, Tinghu Zhang, Sirano Dhe-Paganon, Jarrod A. Marto,* and Nathanael S. Gray*

Cite This: https://doi.org/10.1021/acsmedchemlett.1c00285

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sı Supporting Information

ABSTRACT: Ligand-directed bioconjugation strategies have been

used for selective protein labeling in live cells or tissue samples in

applications such as live-cell imaging. Here we hypothesized that a

similar strategy could be used for targeted protein degradation. To

test this possibility, we developed a series of CDK2-targeting N-

acyl-N-alkylsulfonamide (NASA)-containing acylation probes. The

probes featured three components: a CDK2 homing ligand, a

CRBN

CRL4

E3 ligase recruiting ligand, and a NASA functionality.

We determined that upon target binding, NASA-mediated reaction

resulted in selective functionalization of Lys89 on puriﬁed or native

CDK2. However, we were unable to observe CDK2 degradation,

which is in contrast to the eﬃcient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis

suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work

demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable eﬃcient protein degradation

remains an open question.

KEYWORDS: Covalent degrader, CDK2, ligand-directed, N-acyl-N-alkylsulfonamide, chemical modiﬁcation

argeted protein degradation is a rapidly developing

research ﬁeld focusing on the discovery and development

nine, lysine, or tyrosine) found in the target protein. One of

the key advantages of covalent binders is their prolonged

duration of action. Recently, several PROTACs featuring either

target recruiting ligands with reactive warheads (e.g., HaloTag7

of agents that can induce selective and rapid degradation of a

−3

protein target.

The most common approach in this space

has been the use of small molecules that induce ternary

complex formation between the target of interest and an E3

ubiquitin ligase, resulting in ubiquitination of the target and its

subsequent proteasome-mediated degradation. Degradation-

based targeting strategies have garnered intense interest

because of several key advantages over the traditional

inhibitor-based approaches, such as demonstrating potential

in cases where a ligand binds directly to the target but has

diﬃculty aﬀecting or blocking the function of the protein.

Unlike “molecular glues” (or monovalent degraders) that

currently lack rational development strategies for new

fusion proteins, ERK1/2, BTK, and KRAS ) or E3

ligase recruiting ligands with reactive warheads (e.g., RNF4,

DCAF16, RNF114, and KEAP1 ) have been reported.

However, the majority of the reported PROTACs degrade

targets traditionally considered as ligandable, leaving the space

of “unligandable” targets poorly explored.

Over the past decade, a range of bio-orthogonal reactions

that enable chemo- and regioselective covalent labeling of

proteins have been developed to enable selective chemical

17,18

modiﬁcation of proteins in their native environment.

These strategies deliver diﬀerent types of probes that can be

used to study protein structure, function, dynamics, and

localization. Recently, ligand-directed N-acyl-N-alkylsulfona-

mide (NASA) chemistry was shown to target nucleophilic

lysine residues located in the proximity of the ligand binding

targets, proteolysis-targeting chimeras (PROTACs), which

are bivalent degraders that include two ligands, one to recruit

the target and the other to recruit the E3 ligase, are amenable

to rational design, as evidenced by rapidly growing list of

targets for which PROTACs have been developed. Most

Received: May 21, 2021

Accepted: July 20, 2021

PROTACs developed to date are degraders that bind to the

target and the E3 ligase reversibly. An orthogonal strategy to

access proteins that are diﬃcult to target through reversible

binding alone is to equip the small-molecule ligand with an

electrophilic warhead to form a covalent bond with a

nucleophilic side-chain residue (e.g., cysteine, serine, threo-

XXXX American Chemical Society

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and red glyphs, respectively. K * denotes TMX-3063-modi ﬁ ed lysine.

Letter

site. In this strategy, the ligand serves as a homing device to

recognize and bind a protein target of interest and deliver a

payload to the proximal lysine via NASA-mediated acylation, as

shown for heat shock protein 90 (HSP90) and human

double minute 2 (HDM2). Because this reaction is rapid and

irreversible, a possible advantage of this strategy is the ability to

covalently label a protein of interest even in the absence of a

tightly bound homing ligand. Building on this, degrader

molecules that use a NASA delivery approach to covalently tag

a protein target with a recruitment ligand for an E3 ligase could

potentially expand the scope of degradable proteome,

especially for unligandable proteins that have protein−protein

interactions with their known ligandable partner proteins.

Here, as a proof of concept, we integrated the NASA approach

into the development of degraders and developed the initial

series of eﬃcient NASA-directed cyclin-dependent kinase 2

(

CDK2) acylation probes. We demonstrated that the probes

eﬀectively labeled Lys89 on CDK2. Notably, we successfully

acylated CDK2 with the degrader compound TMX-3054 and

examined the consequences of this modiﬁcation in living cells.

In summary, TMX-3054 can eﬃciently engage native CDK2

with high site selectivity, but it failed to induce productive

binary complex formation between acylated CDK2 and

cereblon (CRBN), potentially explaining why we failed to

observe eﬀective degradation of CDK2 in OVCAR8 cells.

Design and Characterization of NASA-Based Acyla-

tion Probes. To identify a suitable proof-of-concept target,

we queried kinome-wide sequence alignments and crystal

structures of human protein kinases. One of our selection

criteria was the presence of a solvent-exposed lysine residue

located near the ATP-binding pocket. On this basis, more than

Figure 1. Mass-spectrometry-based analyses demonstrated that TMX-

063 reacted with CDK2 protein at Lys89. (A) Schematic illustration

of covalent modiﬁcation of CDK2 by TMX-3063. (B) CDK2

inhibitory activities of TMX-3063 and TMX-3066. (C) Mass spectra

(

left) and zero-charge mass spectra (right) of CDK2 protein treated

with DMSO (top) or a 2-fold molar excess of TMX-3063 (bottom)

for 1 h at room temperature. The observed mass shift of 42 Da is

consistent with covalent addition of a single molecule of TMX-3063

(

with loss of the TMX-3066 fragment). (D) CE−MS/MS spectrum

0 kinases met our criteria (Table S1 ), among which Lys89 on

of chymotryptic CDK2 peptide (residues 89 to 104) with Lys89

modiﬁed by TMX-3063. Ions of type b and y are indicated with blue

CDK2 emerged as a top hit particularly because it was

previously targeted by the covalent CDK2 inhibitor NU6300.

Moreover, we recently described the non-covalent picomolar

CDK2 inhibitor TMX-3013, which contains a sulfonamide

functionality suitable for installation of a NASA warhead. Thus,

we used TMX-3013 as the CDK2 homing ligand and

introduced the NASA warhead at the 4-position of the phenyl

ring, resulting in the generation of TMX-3063, which displayed

potent inhibitory activity against CDK2/cyclin A with an IC₅₀

value of 2.2 nM as measured using a commercially available

CDK2 biochemical assay (Figure 1A,B and Table S2 ). To

examine our hypothesis that TMX-3063 would perform ligand-

directed acetylation of the ε-amino group of Lys89, we ﬁrst

used liquid chromatography−mass spectrometry (LC−MS) to

analyze the recombinant CDK2 protein incubated with a 2-fold

molar excess of TMX-3063 for 1 h at room temperature. As

expected, a mass shift consistent with the stoichiometric

acetylation of CDK2 was observed (Figure 1C). To determine

the site of modiﬁcation, the labeled protein was digested with

trypsin, and the peptides were analyzed by capillary electro-

phoresis−mass spectrometry (CE−MS). A database search

revealed exclusive modiﬁcation of Lys89 (Figure 1D). Taken

together, our mass-spectrometry-based analyses conﬁrmed that

TMX-3063 is a NASA-based acylation reagent that acetylates

Lys89 on CDK2.

Figure 2. TMX-3063 labels CDK2 in cells. (A) Chemical structure of

TMX-3052 and its CDK2/cyclin A inhibitory activity. (B) OVCAR8

cells were pretreated with either DMSO or TMX-3063 (1 μM) for 4 h

followed by cell lysis, and the cell lysates were then incubated with

TMX-3052 (1 μM) for 16 h. The protein was denatured with 4 mM

urea + 1% SDS to eliminate the reversible binding.

DMSO or TMX-3063 at a concentration of 1 μM for 4 h, and

the cell lysates were then incubated with desthiobiotin-

conjugated TMX-3052 (1 μM) for 16 h at 4 °C. Protein

bound by TMX-3052 was aﬃnity-puriﬁed using streptavidin

beads and analyzed by sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) and Western blotting. As

shown in Figure 2B, the desthiobiotinylated CDK2 band was

clearly observed regardless of the denaturing condition (lanes 2

and 3), consistent with covalent modiﬁcation of CDK2.

Pretreatment of the cells with TMX-3063 resulted in

protection of CDK2 from pull-down by the desthiobiotin

compound (lanes 4 and 5). Taken together, these results

conﬁrmed cellular target engagement of CDK2 by TMX-3063.

Similar experiments revealed that TMX-3063 can also engage

Next, to determine whether TMX-3063 engages CDK2 in a

cellular environment, we performed competitive pull-down

studies. To enable this experiment, we synthesized TMX-3052

(Figure 2A), a NASA-linked, desthiobiotin-conjugated com-

pound that retains potency as a CDK2 inhibitor. To investigate

cellular target engagement, we treated OVCAR8 cells with

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, four CRBN-recruiting molecules with PEG linkers of various

Letter

CDK1 and CDK5, which also possess a lysine analogous to

Lys89 of CDK2 (Figure S1 ).

Design and Evaluation of NASA-Based Covalent

Probes for CDK2 Degradation. Prior to this study, we

and others have successfully developed PROTAC-based CDK2

degraders, including TMX-2172, which incorporates CRBN-

2,23

recruiting ligands.

degrader molecules based on TMX-3063. As shown in Figure

We designed a series of NASA-directed

Figure 3. Schematic illustration of ligand-directed delivery of a

CRBN-recruiting tag onto Lys89 of CDK2.

Figure 4. Mass spectrometry analysis and an X-ray crystallography

study revealed that TMX-3054 reacted with CDK2 protein at Lys89.

lengths (TMX-3054, TMX-3166, TMX-3164, and TMX-3185)

were synthesized. Biochemical activity tests conﬁrmed that all

of these compounds were potent CDK2 inhibitors with IC₅₀

values below 20 nM (Table S2). Next, to determine whether

these molecules could also form a covalent bond with Lys89 of

recombinant CDK2 protein, we performed MS labeling studies

using TMX-3054 as a representative. A mass shift consistent

with ca. 75% modiﬁcation of CDK2 protein was observed after

incubation for 2 h at room temperature, and subsequent CE−

MS analysis of chymotryptic peptides conﬁrmed the site of

modiﬁcation (Figure 4A,B). To visualize the CRBN-recruiting

ligand-tagged structure, recombinant CDK2 was cocrystallized

in complex with TMX-3054, and a structure was determined to

a resolution of 3.08 Å. We observed electron density in the

ATP-binding pocket consistent with the chemical structure of

TMX-3066, which is the expected product following reaction

of TMX-3054 with Lys89 of CDK2. We were unable to

observe any electron density for the CRBN-recruiting tag on

Lys89, presumably because it was solvent-exposed and

disordered (Figure 4C).

(

A) Mass spectra (left) and zero-charge mass spectra (right) of CDK2

protein treated with DMSO (top) or a 10-fold molar excess of TMX-

054 (bottom) for 2 h at room temperature. The observed mass shift

of 561 Da is consistent with covalent addition of a single molecule of

TMX-3054 (with loss of the TMX-3066 fragment). (B) CE−MS/MS

spectrum of chymotryptic CDK2 peptide (residues 81 to 90) with

Lys89 modiﬁed by TMX-3054. Ions of type b and y are indicated with

blue and red glyphs, respectively. K* denotes TMX-3054-modiﬁed

lysine. (C) Cocrystal structure of CDK2 (3.08 Å) in complex with

TMX-3054 (PDB ID 7MKX). The electron density of the released

bound ligand TMX-3066 is colored in clay. The electron density of

Lys89 (shown as gray sticks) was present, but the linked CRBN-

recruiting moiety was not and was presumed to be completely

solvent-exposed and disordered.

compounds (Figure S2 ). As shown in Figure 6A, no CDK2

degradation was observed upon treatment with TMX-3054 in

the concentration range of 10 nM−10 μM. To rule out the

possibility of poor cell permeability, a cellular target pull-down

study was performed. Similar to the results for TMX-3063, as

shown in lanes 4 and 5 of Figure 6B, the biotinylated CDK2

band was signiﬁcantly attenuated after 4 h treatment of

OVCAR8 cells with TMX-3054 at a concentration of 1 μM,

indicating eﬃcient target engagement on CDK2 (the cellular

engagement data for TMX-3054 with CDK5 and CDK1 are

available in Figure S1 ). Taken together, these results indicated

that CDK2 is fully modiﬁed with a CRBN-recruiting tag on

Lys89 via NASA-based molecules. However, the modiﬁed

CDK2 is unable to bind CRBN and is therefore not targeted

for degradation.

Targeted protein degradation is one of the most rapidly

growing areas of research within chemical biology and drug

discovery. In addition to reversible bivalent degraders that are

commonly pursued, covalent bivalent degraders provide an

alternative way to access proteins that are diﬃcult to target

through reversible binding alone. Although successful examples

of covalent bivalent degraders either covalently binding target

To investigate whether any of the NASA-based compounds

could degrade CDK2, we generated the CDK2 HiBit assay,

which enabled quantitative measurement of CDK2 protein

levels in living cells. As shown in Figure 5A, none of the

CRBN-recruiting-tag molecules resulted in signiﬁcant reduc-

tion of the HiBit signals in the concentration range of 0.1−10

μM. In contrast, our previously developed reversible bivalent

degrader TMX-2172 signiﬁcantly reduced the signal, indicating

eﬃcient degradation of HiBit−CDK2. To examine whether the

lack of degradation in the presence of covalently attached

CRBN-recruiting tag was due to the inability to bind CRBN or

the failure to form a productive binary complex, we generated a

NanoBRET assay. Surprisingly, none of the NASA-based

molecules produced a ﬂuorescence signal following incubation

of cells at concentrations up to 30 μM (Figure 5B). This was

cross-validated by a negative result from immunoblot analysis

of native CDK2 in OVCAR8 cells after 6 h treatment with the

ACS Medicinal Chemistry Letters

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The Supporting Information is available free of charge at

Letter

orientation of the tag as well as the target selection, appear to

play a major role, similar to what has been seen for reversible

bivalent degraders. Collectively, our results indicate that

further work is needed to establish the most eﬀective way to

rapidly optimize NASA-based acylation chemistry toward

achieving degradation within and beyond kinase family of

proteins.

ASSOCIATED CONTENT

sı Supporting Information

■

https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00285.

Biological activities, assay protocols, synthetic proce-

dures, and compound characterization data (PDF)

AUTHOR INFORMATION

Corresponding Authors

■

Jarrod A. Marto − Department of Cancer Biology and

Department of Oncologic Pathology and Blais Proteomics

Center, Dana-Farber Cancer Institute, Boston, Massachusetts

02115, United States; Department of Pathology, Brigham

and Women’s Hospital, Harvard Medical School, Boston,

Massachusetts 02115, United States; orcid.org/0000-

Figure 5. HiBiT (top) and NanoBRET (bottom) target engagement

with degraders. (A) HiBiT, which complements with LgBiT to form

the luminescence, was inserted via CRISPR/Cas9 to the C-terminus

of CDK2 in HEK293T cells. Following treatment with degraders,

protein degradation was monitored by the loss of luminescence. (B)

In HEK293T cells, NanoLuc−CDK2 was used as the energy donor

and HaloTag−CRBN was used as the energy acceptor. Following

treatment with degraders, the CDK2:CRBN complex formation was

monitored by the NanoBRET ratio (acceptor emission at 618 nm/

donor emission at 460 nm).

0003-2086-1134 ; Email: Jarrod_Marto@dfci.harvard.edu

Nathanael S. Gray − Department of Chemical and Systems

Biology, ChEM-H, Stanford Cancer Institute, School of

Medicine, Stanford University, Stanford, California 94305,

United States; orcid.org/0000-0001-5354-7403;

Email: nsgray01@stanford.edu

Authors

Mingxing Teng − Department of Cancer Biology, Dana-

Farber Cancer Institute, Boston, Massachusetts 02115,

United States; Department of Biological Chemistry and

Molecular Pharmacology, Harvard Medical School, Boston,

Massachusetts 02115, United States; orcid.org/0000-

0001-6574-1764

Jie Jiang − Department of Cancer Biology, Dana-Farber

Cancer Institute, Boston, Massachusetts 02115, United

States; Department of Biological Chemistry and Molecular

Pharmacology, Harvard Medical School, Boston,

Figure 6. Immunoblot analysis of CDK2 in OVCAR8 cells. (A)

OVCAR8 cells were treated with TMX-3054 for 6 h in the

concentration range of 10 nM−10 μM. (B) OVCAR8 cells were

pretreated with either DMSO or TMX-3054 (1 μM) for 4 h followed

by cell lysis, and then the cell lysates were incubated with TMX-3052

Massachusetts 02115, United States

Scott B. Ficarro − Department of Cancer Biology and

Department of Oncologic Pathology and Blais Proteomics

Center, Dana-Farber Cancer Institute, Boston, Massachusetts

(

1 μM) for 16 h.

2115, United States; Department of Pathology, Brigham

proteins or covalently binding E3 ligases have been reported,

degrading proteins that lack a small-molecule binding pocket

are considered challenging. Here we explored the possibility of

using proximity-driven, ligand-directed reaction to label a

target protein with an E3 recruiting ligand as a simple

degradation tag. With CDK2 as a model system, our probes

successfully labeled the native CDK2 with a CRBN-recruiting

tag on Lys89. However, we did not observe CDK2 degradation

under the conditions tested, and our follow-up analysis

suggests that the likely reason for this is the failure of

productive CDK2:CRBN complex formation. Thus, our work

demonstrates that NASA-based chemistry can be an eﬀective

approach for installing an E3 ligase recruiting tag on the target

of interest. However, our work also shows that the presence of

the recruiting tag is insuﬃcient to ensure target degradation

and that additional factors, such as the exact location and

and Women’s Hospital, Harvard Medical School, Boston,

Massachusetts 02115, United States

Hyuk-Soo Seo − Department of Cancer Biology, Dana-Farber

Cancer Institute, Boston, Massachusetts 02115, United

States; Department of Biological Chemistry and Molecular

Pharmacology, Harvard Medical School, Boston,

Massachusetts 02115, United States; orcid.org/0000-

0003-0646-2102

Jae Hyun Bae − Department of Cancer Biology, Dana-Farber

Cancer Institute, Boston, Massachusetts 02115, United States

Katherine A. Donovan − Department of Cancer Biology,

Dana-Farber Cancer Institute, Boston, Massachusetts 02115,

United States; Department of Biological Chemistry and

Molecular Pharmacology, Harvard Medical School, Boston,

Massachusetts 02115, United States; orcid.org/0000-

0002-8539-5106

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

Letter

Eric S. Fischer − Department of Cancer Biology, Dana-Farber

Cancer Institute, Boston, Massachusetts 02115, United

States; Department of Biological Chemistry and Molecular

Pharmacology, Harvard Medical School, Boston,

mass spectrometry; SDS-PAGE, sodium dodecyl sulfate

polyacrylamide gel electrophoresis

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Massachusetts 02115, United States; orcid.org/0000-

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ACKNOWLEDGMENTS

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ABBREVIATIONS

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modification. Nat. Commun. 2014, 5, 4740.

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Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid labelling and covalent

CDK2, cyclin-dependent kinase 2; NASA, N-acyl-N-alkylsulfo-

namide; CRBN, cereblon; CE−MS, capillary electrophoresis−

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