bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022.

The copyright holder for this preprint

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Selective inhibitors of JAK1 targeting a subtype-restricted allosteric cysteine

Madeline E. Kavanagh1,2, Benjamin D. Horning1,3, Roli Khattri3, Nilotpal Roy3, Justine P. Lu3,
Landon R. Whitby3, Jaclyn C. Brannon3, Albert Parker3, Joel M. Chick3, Christie L. Eissler3,
Ashley Wong3, Joe L. Rodriguez3, Socorro Rodiles3, Kim Masuda2, John R. Teijaro4, Gabriel M.
Simon3, Matthew P. Patricelli3*, Benjamin F. Cravatt2*
1
These authors contributed equally, 2Department of Chemistry, Scripps Research, La Jolla, CA

92037, USA; 3Vividion Therapeutics, 5820 Nancy Ridge Drive, San Diego, CA 92121, USA;
4
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla,

CA, 92307, USA.

*Correspondence – cravatt@scripps.edu; mattp@vividion.com

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Abstract

The JAK family of non-receptor tyrosine kinases includes four subtypes (JAK1, JAK2, JAK3,

and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors.

JAK inhibitors have emerged as important therapies for immuno(onc)ological disorders, but their

use is limited by undesirable side effects presumed to arise from poor subtype selectivity, a

common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here, we describe

the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic

pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3.

Electrophilic compounds selectively engaging this site block JAK1-dependent

transphosphorylation and cytokine signaling, while appearing to act largely as “silent” ligands for

TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine

signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an

allosteric approach for inhibiting JAK1 with unprecedented subtype selectivity.

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available under aCC-BY-NC-ND 4.0 International license.

Dysregulated cytokine signaling is central to the pathophysiology of a wide range of diseases,

including autoimmune and inflammatory conditions, cardiovascular, gastrointestinal and

neurodegenerative diseases, and cancer1,2. More than 50 different cytokines signal through a

family of non-receptor Janus tyrosine kinases (JAKs), which, in humans, consists of JAK1,

JAK2, JAK3, and TYK21,3. JAKs associate with the intracellular tail of specific cytokine receptors

and are activated by receptor-induced dimerization to phosphorylate themselves in trans, the

receptor, and downstream signaling proteins, including the STAT family of transcription factors.

The specific combination of JAK enzymes and STAT transcription factors that are activated by a

given cytokine is cell-type and context-dependent, allowing the JAK-STAT system to regulate

diverse biological and disease processes3.

The key role of JAK-STAT pathways in immunology and cancer has motivated the

pursuit of JAK inhibitors, and many pan-JAK inhibitors have been described1,4. These

compounds have provided preclinical and clinical evidence that inhibiting JAK-STAT signaling

can alleviate aberrant cytokine responses and have established JAKs as important therapeutic

targets1,4. There are currently seven FDA-approved JAK inhibitors for the treatment of

rheumatoid arthritis, psoriasis, ulcerative colitis, atopic dermatitis, and/or myeloproliferative

diseases (e.g. polycythemia, leukemia and GVHD), and one compound (baracitinib) that has

emergency use authorization (EUA) for COVID-194. All FDA-approved JAK inhibitors act by an

orthosteric mechanism, meaning that they bind to the conserved ATP pocket of the kinase

domain, and, even though individual compounds have differing relative selectivity profiles

across the JAK family, they all inhibit more than one JAK isoform with moderate-to-high potency

(IC50 < 1 μM)5–12. This lack of selectivity has important translational implications, as there is

growing concern over an array of adverse side effects caused by JAK inhibitors1,3,4,13, including

dose-limiting cytopenias thought to be due to inhibition of JAK2-mediated growth factor receptor

signaling14, an increased risk of cardiovascular events and infections, dyslipidemia, and

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elevated liver enzymes3. In 2021, these concerns prompted the FDA to place a “black box”

warning on JAK inhibitors indicated for chronic conditions such as rheumatoid arthritis15.

The lack of subtype selectivity of FDA-approved JAK inhibitors may contribute to their

adverse clinical effects16,17, and there is accordingly considerable interest in the discovery of

JAK inhibitors with improved specificity. Such compounds might not only constitute next-

generation therapeutics, but would also serve as valuable research tools to better understand

the unique contributions made by each JAK isoform to physiology and disease. Subtype-

selective JAK inhibitors have been pursued by multiple strategies. For example, covalent

inhibitors of JAK3 have been developed, such as ritlecitinib, that target a cysteine (C909)

uniquely found in the activation loop of this kinase compared to other JAKs18,19. While this

approach achieves specificity for JAK3 over other JAKs, ritlecitinib cross-reacts with TEC family

kinases, which also share a cysteine at an equivalent position. JAKs are distinguished from

many other kinases by having an additional non-catalytic pseudokinase (JH2) domain that

regulates kinase activity and is a hotspot for gain- or loss-of-function mutations2,20. Notably,

compounds binding to the ATP pocket of the JH2 domain of TYK2 have been found to inhibit

this kinase with remarkable functional selectivity over JAK1-JAK321,22, and one of these agents,

– BMS-986165 (deucravacitinib) – is in late-stage clinical development for autoimmune

disorders21,23,24.

In contrast to the progress made on subtype-restricted JAK3 and TYK2 inhibitors,

selective JAK1 inhibitors are still lacking. Although some orthosteric JAK1 inhibitors have been

reported that display improved subtype selectivity, these compounds (e.g., abrocitinib, filgotinib)

still generally show substantial cross-reactivity with JAK2 (e.g., < 1 µM IC50 values), depending

on the biochemical or cellular assay employed12,25,26. The generation of highly selective

inhibitors of JAK1 is an important objective, as several lines of evidence indicate that JAK1

blockade contributes to the efficacy of pan-JAK inhibitors in chronic autoimmune disorders. For

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instance, gain-of-function JAK1 mutations promote multi-organ immune dysregulation27 and are

associated with specific types of cancer (e.g., leukemia28,29 and gynecological tumors30), while

deleterious mutations cause severe immunosuppression in humans31. Additionally, JAK1 is

broadly expressed and plays essential and non-redundant roles downstream of class II, γc, and

gp130 cytokines32, many of which are dysregulated in inflammatory diseases2. Nonetheless, the

precise contribution of JAK1 to homeostatic immune function and disease remains only partly

understood due to a lack of genetic models and selective chemical tools. JAK1 deletion is

perinatal lethal to mice32, and consequently, much of our understanding of JAK1 biology has

relied on studies with conditional knockout mice lacking JAK1 in specific cell types33–35, JAK1-

deficient human cell lines20,36–39 and/or non-selective orthosteric inhibitors16,36,40.

Here, we describe the chemical proteomic discovery of a ligandable allosteric cysteine in

the pseudokinase domain of JAK1 (C817) and TYK2 (C838) but absent from JAK2 and JAK3.

We optimize an electrophilic compound VVD-118313 (5a) that engages JAK1_C817 and

TYK2_838 with high potency and proteome-wide selectivity and show that this agent blocks

JAK1 signaling in human cancer cell lines and primary immune cells, while sparing JAK2-

dependent pathways. VVD-118313 does not inhibit signaling of a C817A JAK1 mutant and

appears to act as a silent ligand for TYK2 in the context of primary immune cells. Mechanistic

studies indicate that VVD-118313 does not inhibit the catalytic activity of purified JAK1, but

potently blocks JAK1 trans-phosphorylation in cells. Integrating our findings with previous work

on allosteric TYK2 inhibitors, such as BMS-986165, points to the potential for leveraging

multiple druggable pockets in the pseudokinase domain of JAKs to develop inhibitors with

unprecedented subtype selectivity.

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Results

Discovery of a ligandable cysteine in the JAK1/TYK2 pseudokinase domain

Previous activity-based protein profiling (ABPP) studies to assess the interactions of

electrophilic small-molecule fragments with cysteines in primary human T cells uncovered a

ligandable cysteine shared by JAK1 (C817) and TYK2 (C838)41. Both cysteines were

substantially engaged by chloroacetamide (KB02) and acrylamide (KB05) fragments (Extended

Data Fig. 1a)41, as determined by mass spectrometry (MS)-ABPP experiments that monitored

electrophile-dependent changes in iodoacetamide-desthiobiotin (IA-DTB) reactivity of > 10,000

cysteines in the human T-cell proteome (Fig. 1a). Other quantified JAK1 and TYK2 cysteines

were unaffected in their IA-DTB reactivity by KB02 or KB05 treatment (Fig. 1b and Extended

Data Fig. 1b).

JAK1_C817 and TYK2_C838 are located in the catalytically inactive pseudokinase (JH2)

domain shared across the JAK family (Fig. 1c). The JH2 domain has been found to regulate the

kinase activity of the JH1 domain through allosteric mechanisms and is a hotspot for gain- or

loss-of-function mutations (Fig. 1c)2,20,42–44. We noted that other JAK family members – JAK2

and JAK3 – did not share the ligandable cysteine (Fig. 1d). A closer examination of the JAK1

JH2 crystal structure in comparison to other kinase structures revealed that C817 is in the C-

lobe proximal to a pocket formed by helices aE-F and aH-I, which, in the structurally related

kinase ABL, binds an auto-inhibitory N-terminal lipid (myristoylation) modification45–47 (Fig. 1e).

This pocket in ABL is targeted by the allosteric inhibitor asciminib, which stabilizes the inactive

conformation of the kinase48 and has recently been approved for the treatment of chronic

myeloid leukemia49. Even though asciminib is a reversible inhibitor, and JAK1 and TYK2 are not

themselves known to be myristoylated (and do not possess an N-terminal Met-Gly sequence

required for myristoylation50), the proximity of C817/C838 to a pocket that has been exploited to

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create allosteric drugs of another kinase encouraged us to further characterize the potential

functional impact of electrophilic compounds targeting these cysteines.

Optimization of covalent allosteric ligands that act as JAK1 inhibitors

We pursued the discovery of more potent and selective covalent ligands for

JAK1_C817/TYK2_C838 by screening an internal library of electrophilic compounds using a

targeted MS-ABPP assay. This approach furnished an attractive piperidine butynamide

fragment hit 1a (Fig. 1f) that showed target engagement values (TE50s) of 2.1 µM and 45 µM for

JAK1_C817 and TYK2_C838, respectively (Fig. 1g and Table 1). Using a homogeneous time-

resolved fluorescence (HTRF) assay in human peripheral blood mononuclear cells (PBMCs),

we also found that 1a inhibited IFNα-stimulated STAT1 phosphorylation – a JAK1/TYK2-

dependent cytokine pathway – with an IC50 value of 1.4 µM (Fig. 1h and Table 1), suggesting

that covalent ligands targeting JAK1_C817/TYK2_C838 acted as JAK1 and/or TYK2 inhibitors.

The corresponding racemate of 1a (compound 1) was ~two-fold less active in both HTRF and

TE assays (Table 1), indicating a stereochemical preference for engagement of

JAK1_C817/TYK2_C838. We next synthesized a focused library of 1a analogues (Fig. 2a) and,

based on the greater potency displayed by 1a for JAK1_C817 over TYK2_C838, screened

these compounds for: i) in vitro engagement of JAK1_C817 (TE50) in human cell proteomes by

targeted MS-ABPP; and ii) cell-based functional activity (IC50) on JAK1-dependent signaling

pathways (IFNα-STAT1, IL-6-STAT3) in human PBMCs. We iteratively improved the potency of

compound interactions with JAK1_C817 by three orders of magnitude and observed a strong

correlation (R2 ~ 0.93-98) between these in vitro TE50 values and the in situ IC50 values for

blocking STAT phosphorylation (Fig. 2b, c and Table 1), further supporting that the compounds

acted as functional antagonists of JAK1. Key structural modifications contributing to improved

potency included the addition of a second chlorine atom at the meta-position of the phenyl ring

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(e.g., compound 3) and replacement of the terminal methyl of the butynamide with a pyrrolidine-

methylsulfonamide (e.g., compound 5). The tested compounds generally showed >10-fold

greater potency for engagement of JAK1_C817 compared to TYK2_C838 (Table 1). Separation

of the stereoisomers of compound 5 revealed that the enantiomers(S, R)-5a and (R, S)-5b were

substantially more potent than the corresponding diastereomers (5c and 5d), blocking STAT

phosphorylation with IC50 values (~0.03-0.05 µM) that were superior to the pan-JAK inhibitor

tofacitinib (Fig. 2c and Table 1). Compound 5a, hereafter referred to as VVD-118313, was

selected for further functional characterization as the compound showed the strongest overall

activity across the assays.

VVD-118313 selectively inhibits JAK1 through engagement of C817

We next evaluated the broader proteomic reactivity of VVD-118313 by untargeted MS-ABPP in

human PBMCs. Across >14,000 quantified cysteines, JAK1_C817 was the most potently

engaged site by VVD-118313 (0.01 – 10 µM, 3h), followed by TYK2_C838, with both cysteines

showing near-complete blockade in their IA-DTB reactivity in cells treated with 0.1 µM of VVD-

118313 (Fig. 2d, e and Supplementary Dataset 1). Two additional cysteines (HMOX2_C282,

SLC66A3_C135) were engaged by VVD-118313 when tested at a 10-fold higher concentration

(1 µM; Fig. 2d, e). Similar results were obtained in MS-ABPP experiments that analyzed the in

vitro proteome-wide reactivity of VVD-118313 in PBMC lysates, where JAK1_C817 was again

the most potently engaged cysteine, followed by TOR4A_C21, a site that was also engaged in

situ, albeit more weakly, and TYK2_C838 (Extended Data Fig. 2 and Supplementary Dataset

1). Taken together, these chemical proteomic data support that VVD-118313 is a highly potent

and selective covalent ligand for JAK1_C817 and TYK2_C838.

To test whether VVD-118313 inhibits JAK1 through engagement of C817, we

recombinantly expressed WT-JAK1 and a C817A-JAK1 mutant in the 22Rv1 human prostate

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cancer cell line, which has a frameshift mutation in the JAK1 gene and consequently only

expresses JAK2 and TYK2 (JAK3 is immune cell-restricted in its expression)3. We also

evaluated a C810A-JAK1 mutant, as the quantified C817 tryptic peptide in our MS-ABPP

experiments also contained C810, a residue that is not conserved in TYK2 (Fig. 1d) and is

further away than C817 from the pocket predicted to bind electrophilic compounds (Fig. 1e). We

first treated 22Rv1 cells expressing the JAK1 variants with an alkynylated analogue of VVD-

118313 (alkyne probe 6 (0.1 µM, 2 h); Fig. 2f) and, after cell lysis, detected 6-labeled proteins

by copper-catalyzed azide-alkyne cycloaddition (CuAAC)51 with a rhodamine (Rh)-azide reporter

group, followed by SDS-PAGE and in-gel fluorescence scanning. Alkyne probe 6 reacted with

WT- and C810A-JAK1, but not C817A-JAK1, and the labeling of WT-JAK1 was blocked in a

concentration-dependent manner by pre-treatment with VVD-118313 (Fig. 2f). We interpret

these data to indicate that VVD-118313 site-specifically engages JAK1 at C817.

JAK1 mediates STAT phosphorylation downstream of different cytokine receptors by

heterodimerizing with other JAK family members (Fig. 3a)3. We selected a representative

subset of these pathways (IFNα-STAT1 and IL-6-STAT3), along with a JAK2-mediated pathway

(prolactin (PRL)-STAT5), to evaluate the functional effects of VVD-118313 on the activity of

recombinantly expressed WT and mutant forms of JAK1 in 22Rv1 cells. We first verified that

recombinant WT-JAK1 and the C810A and C817A mutants equivalently rectified intrinsic

defects in IFNα and IL-6 signaling in parental 22Rv1 cells37,52, as reflected by the greater IFNα

or IL-6-stimulated STAT1/3 phosphorylation in cells expressing these JAK1 variants compared

to mock cells (Fig. 3b and Extended Data Fig. 3a). We also noted that all of the JAK1 variant-

expressing cells displayed a similar degree of constitutive phosphorylation of the JAK1 JH1

activation loop (Y1034/Y1035) that was not further increased by cytokine treatment (Fig. 3b).

VVD-118313 (2 µM, 2 h) blocked IFNα-simulated STAT1 and IL-6-stimulated STAT3

phosphorylation in WT- or C810A-JAK1-expressing 22Rv1 cells, but not in C817A-JAK1-

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expressing cells (Fig. 3b, c). In contrast, the orthosteric JAK inhibitor tofacitinib equivalently

blocked IFNα-simulated STAT1 phosphorylation in cells expressing WT-, C810A-, or C817A-

JAK1 (Fig. 3b, c). Interestingly, VVD-118313 also completely blocked the constitutive

phosphorylation of WT- and C810A-JAK1 but did not affect the phosphorylation of C817A-

JAK1(Fig. 3b, d). In contrast, tofacitinib only partly (~50%) reduced phosphorylation of all JAK1

variants (Fig. 3b, d). VVD-118313 and tofacitinib further differed in their effects on JAK2-

mediated signaling, where VVD-118313 was inactive, while tofacitinib fully inhibited PRL-

induced STAT5 phosphorylation (Fig. 3b, c). Concentration-dependent analyses revealed that

VVD-118313 maximally inhibited IFNα-STAT1 and IL-6-STAT3 phosphorylation (> 80% in each

case) in WT- or C810A-JAK1-expressing 22Rv1 cells at ~0.2 µM, while showing negligible

impact (< 10%) in cells expressing C817A-JAK1 up to 2 µM (Fig. 3e and Extended Data Fig.

3b, c). VVD-118313 inhibited WT- and C810A-JAK1 phosphorylation with even greater potency

than STAT1/STAT3 phosphorylation, showing maximal activity (> 90% blockade) at 0.05 µM

(Fig. 3e), which could indicate that only a small fraction of residually phosphorylated and

activated recombinant JAK1 is required to support signal transduction in IFNα/IL-6-stimulated

22Rv1 cells. Together, these data indicate that VVD-118313 acts as a selective allosteric

inhibitor of JAK1 through covalent engagement of C817.

To determine if VVD-118313 also inhibited TYK2 signaling, we first confirmed that

alkyne probe 6 reacted with recombinantly expressed WT-TYK2, but not a C838A-TYK2 mutant,

in 22Rv1 cells, and that pre-treatment with VVD-118313 blocked compound 6 reactivity with WT

TYK2 (Extended Data Fig. 4a). We did not observe a signal for endogenous TYK2 in these

experiments (e.g., in mock 22Rv1 cells), indicating that the expression level of TYK2 in 22Rv1

cells was too low to visualize with alkyne probe 6. 22Rv1 cells expressing recombinant WT- or

C838A-TYK2 displayed modestly increased IFNα-induced STAT1 phosphorylation compared to

mock 22Rv1 cells (Extended Data Fig. 4b), suggesting that atypical TYK2 homodimers and/or

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TYK2-JAK2 heterodimers can partly support IFNα signaling in these cells37,52,53. VVD-118313

(0.01 - 5 μM, 2 h) inhibited IFNα-STAT1 phosphorylation in 22Rv1 cells expressing WT-TYK2,

but not C838A-TYK2, while the TYK2 inhibitor BMS-986165 blocked IFNα-STAT1

phosphorylation in both cell populations (Fig. 3f). We further noted that VVD-118313 and BMS-

986165 blocked the weaker IFNα/IL-6-stimulated STAT1/STAT3 signaling in mock 22Rv1 cells,

which lack JAK1 expression (Extended Data Fig. 4c-f) suggesting that these pathways are

mediated by endogenous TYK2. Similar to what we observed for JAK1, VVD-118313 inhibited

phosphorylation of the activation loop of WT-, but not C838A-TYK2 (Fig. 3f). BMS-986165 also

suppressed TYK2 phosphorylation; however, this effect was independent of C838 (Fig. 3f).

These data support that VVD-118313 can site-specifically inhibit the signaling of TYK2, at least

in the context of a cell line where JAK1 is absent.

VVD-118313 selectively inhibits JAK1 signaling in primary immune cells

We next evaluated the activity of VVD-118313 (5a) and its mixture of stereoisomers (compound

5) in primary human immune cells and found that both compounds inhibited IFNα-pSTAT1, IL-6-

pSTAT3, and IL-2-pSTAT5 pathways in a concentration-dependent manner (Fig. 4a-c). At

concentrations of 0.1 and 1 µM - where VVD-118313 fully engaged JAK1_C817 in human

PBMCs (Fig. 2d, e) and substantially blocked IFNα, IL-6, and IL-2 signaling (Fig. 4a-c) - no

effects on GM-CSF/JAK2-mediated STAT5 phosphorylation were observed (Fig. 4d). When

tested at 10 µM, VVD-118313 showed modest inhibitory effects on GM-CSF/JAK2-STAT5

phosphorylation (Fig. 4d). Considering that 10 µM of VVD-118313 is a 100-fold greater

concentration than that required to fully engage JAK1_C817, we interpret the modest inhibitory

activity on GM-CSF/JAK2-STAT5 phosphorylation to reflect an off-target activity and note that

several cysteines in the human PBMC proteome were substantially engaged by this compound

at 10 µM (Extended Data Fig. 5a and Supplementary Dataset 1). VVD-118313 showed the

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strongest inhibitory effect on IFNα-stimulated STAT1 phosphorylation (>50% and >90%

inhibition at 0.01 and 0.1 µM, respectively), followed by IL-6-pSTAT3 (~80-85% inhibition at 0.1-

1 µM) and IL-2-pSTAT5 (~70% inhibition at 0.1-1 µM) pathways. Compound 5 behaved similarly

to VVD-118313, with the expected reduction in potency (Fig. 4a-d) that is in accordance with

TE50 values measured by MS-ABPP (Table 1). The pan-JAK inhibitor tofacitinib blocked all of

the evaluated cytokine-JAK/STAT pathways at a concentration of 1-2 µM (Fig. 4a-d). Finally, we

found that VVD-118313 did not block TYK2-dependent IL-12-STAT4 signaling in human PBMC-

derived T-blasts, which was inhibited by both BMS-986165 and tofacitinib (Fig. 4e). This result

differed from the inhibitory activity displayed by VVD-118313 in JAK1-null 22Rv1 cells, where

the compound suppressed TYK2-dependent STAT1 phosphorylation and suggests that under

more physiological settings, VVD-118313 does not act as a functional antagonist of TYK2.

We were also interested in evaluating whether the covalent allosteric inhibitors were

capable of engaging and inhibiting JAK1 in vivo. We first confirmed by MS-ABPP that VVD-

118313 selectively and completely engaged C816 of mouse JAK1 (the corresponding residue to

human JAK1_C817) in splenocyte proteomic lysates at concentrations as low at 0.01 µM

(Extended Data Fig. 5b and Supplementary Dataset 1). Across >11,000 quantified sites, only

a single additional cysteine BACH1_C438 was engaged > 70% by 1 µM of VVD-118313. Mouse

TYK2 was not targeted by VVD-118313 because this protein possesses a serine residue (S858)

in the position corresponding to human TYK2_C838. As we observed in human immune cells,

VVD-118313 and compound 5 inhibited both IFNa-dependent STAT1 and IL-2-dependent

STAT5 phosphorylation in mouse splenocytes at concentrations as low at 0.01-0.1 µM

(Extended Data Fig. 6a, b). In contrast, VVD-118313 produced only minimal effects (< 30%) on

GM-CSF-STAT5 or IL-12-STAT4 phosphorylation signaling up to 2 µM test concentration

(Extended Data Fig. 6c, d). One unexpected observation was that VVD-118313 did not inhibit

IL-6-STAT3 signaling in mouse splenocytes (Extended Data Fig. 6e), which contrasted with the

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robust inhibition of this pathway observed in human PBMCs (Fig. 4b). Since tofacitinib retained

inhibitory activity against IL-6-STAT3 signaling in mouse splenocytes (Extended Data Fig. 6e,

h), we speculate that this cytokine pathway may be regulated by different JAK subtypes in

human PBMCs versus mouse splenocytes. In support of this hypothesis, we found that BMS-

986165 suppressed IL-6-stimulated STAT3 phosphorylation in mouse splenocytes to an even

greater degree than the pan-JAK inhibitors tofacitinib and upadacitinib (Extended Data Fig. 6f),

suggesting that TYK2 may contribute more substantially than JAK1 to IL-6-stimulated STAT3

phosphorylation in mouse splenocytes.

We next performed in vivo studies using compound 5, which was chosen over VVD-

118313 due to the comparative ease of scaled-up synthesis and the comparable functional

activity of the mixture of stereoisomers in primary immune cells (Fig. 4a-d). Initial

pharmacokinetic studies revealed that compound 5 exhibited a short half-life (0.36 h) and rapid

clearance in mice (112 mL/min/kg) (Extended Data Table 1). Nonetheless, we hypothesized

that the covalent mechanism of action of 5 may overcome its suboptimal pharmacokinetic

properties to still allow for substantial engagement of JAK1_816 in vivo. The compound was

administered subcutaneously to mice at 25 or 50 mg/kg (or vehicle control) in a two-dose

protocol with a 4 h interval between doses. At 4 h after the second dose, mice were sacrificed

and spleen tissue analyzed by targeted MS-ABPP, which confirmed ~75% engagement of

JAK1_C816 at both 25 and 50 mg/kg of compound 5, while other cysteines in JAK1 were

unaffected (Fig. 4f, g). We also found that splenocytes from compound 5-treated mice showed

substantial impairments in IFNa-dependent STAT1 phosphorylation compared to splenocytes

from vehicle-treated mice (Fig. 4h and Extended Data Fig 7). In contrast, IL-2-dependent

STAT5 phosphorylation, which was incompletely blocked by VVD-118313 or compound 5 in

cultured human (Fig. 4c) and mouse (Extended Data Fig. 6b) immune cells, was not

substantially altered in splenocytes from compound 5-treated mice (Fig. 4h and Extended Data

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Fig 7), suggesting that insufficient JAK1 engagement occurred in vivo to impact this pathway.

Finally, consistent with our cultured immune cell studies, IL-6-STAT3 and GM-CSF-STAT5

signaling were unaffected in splenocytes isolated from compound 5-treated mice (Fig. 4h and

Extended Data Fig 7).

Taken together, our data indicate that covalent ligands engaging human JAK1_C817 (or

mouse JAK1_C816) selectively disrupt JAK1-dependent cytokine signaling in human and

mouse immune cells and can serve as chemical probes for both cellular and in vivo studies.

VVD-118313 blocks JAK1 transphosphorylation in a C817-dependent manner

The more extensive blockade of JAK1 phosphorylation by covalent allosteric inhibitors

compared to orthosteric inhibitors (Fig. 3c) pointed to distinct mechanisms of action for each

class of compounds. Also consistent with this premise, VVD-118313 did not inhibit the catalytic

activity of recombinant purified JAK1 (aa 438 - 1154, J01-11G, SignalChem) in a peptide

substrate assay (VA7207, Promega), whereas tofacitinib displayed a strong inhibitory effect

(Fig. 5a). We next explored the potential mechanistic basis for VVD-118313 blockade of JAK1

phosphorylation by evaluating this compound in 22Rv1 cells co-expressing differentially epitope-

tagged catalytically active (FLAG-tagged WT or C817A) or inactive (HA-tagged K908E or

C817A/K908E) variants of JAK1. We first found that, in the absence of VVD-118313, WT-JAK1-

FLAG and C817A-JAK1-FLAG were robustly auto-phosphorylated when individually expressed

in 22Rv1 cells, while the K908E-JAK1-HA and C817A/K908E-JAK1-HA variants did not show

evidence of phosphorylation (Fig. 5b). Co-expression with either catalytically active JAK1-FLAG

variant (WT or C817A) led to clear trans-phosphorylation of either inactive JAK1-HA variant

(K908E or C817A/K908E), although the magnitude of this trans-phosphorylation activity was

noticeably higher in cells expression WT-JAK1-FLAG versus C817A-JAK1-FLAG and weakest

in cells co-expressing both C817A-JAK1-FLAG and C817A/K908E-JAK1-HA (Fig. 5b, c). VVD-

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

118313 (2 µM, 2 h) completely blocked trans-phosphorylation of either inactive JAK1-HA variant

(K908E or C817A/K908E) in cells expressing active WT-JAK1-FLAG, but did not affect trans-

phosphorylation of inactive JAK1-HA variants in cells expressing active C817A-JAK1-FLAG

(Fig. 5b, d). We also found that BMS-986165, which has been shown to bind the JAK1 JH2

domain in vitro (IC50 ~ 1 nM) and inhibit JAK1-dependent cytokine signaling (IL-6 and IL-2) with

an IC50 of ~ 0.5 µM21, blocked JAK1 trans-phosphorylation in a C817-independent manner

(Extended Data Fig. 8a). We interpret these data to indicate that the inhibition of trans-

phosphorylation of JAK1 contributes to the allosteric mechanism of action of VVD-118313 and

that this effect required the presence of C817 on the donor (phosphorylating), but not the

recipient (phosphorylated) JAK1 variant. That the C817A mutation, on its own, partly

suppressed trans-phosphorylation (Fig. 5b, c), despite not impairing cytokine-induced STAT

phosphorylation (Fig. 3c) can be further interpreted to suggest: i) the potential for allosteric

communication between the C817 pocket of the JH2 domain and the JH1 kinase domain of

JAK1, even in the absence of exogenous inhibitor; and ii) only a modest fraction of

phosphorylated JAK1 is required to fully support cytokine-dependent signaling in the 22Rv1 cell

system, as noted above (Fig. 3b-e).

VVD-118313 shows a distinct functional selectivity profile among JAK inhibitors

In addition to supporting cytokine signaling through phosphorylation of downstream substrates

(e.g., STATs), JAK kinases have also been found to play scaffolding roles in these signal

transduction pathways. For instance, both JAK1 and JAK2 participate in the IFNγ-STAT1

pathway, but only the catalytic activity of JAK2 is required for STAT1 phosphorylation, while the

JAK1 pseudokinase domain is thought to serve a scaffolding function31,54. We confirmed that

IFNγ promoted STAT1 phosphorylation in 22Rv1 cells expressing recombinant WT-, K908E-, or

C817A/K908E-JAK1, but not in mock-transfected cells (Fig. 5e). In contrast, IFNα-stimulated

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

STAT1 phosphorylation was only supported by catalytically active WT-JAK1, but not the K908E

or C817A/K908E mutants (Fig. 5e). We found that VVD-118313 produced an ~40% partial

blockade of IFNγ-stimulated STAT1 in K908E-JAK1-expressing 22Rv1 cells, but not in

C817A/K908E JAK1-expressing cells (Fig. 5f). In contrast, tofacitinib (1 μM, 2h) fully inhibited

IFNγ-stimulated STAT1 phosphorylation in 22Rv1 cells expressing either K908E-JAK1 or

C817A/K908E-JAK1 (Fig. 5f), which presumably reflects the blockade of endogenous JAK2

activity by this pan-JAK inhibitor. We further evaluated the impact of VVD-118313 on JAK1

scaffolding function in primary human immune cells. We specifically compared the

concentration-dependent effects of VVD-118313 and the JAK1/JAK2 inhibitor upadacitinib10,55

on IFNα-STAT1, IFNγ-STAT1, and GM-CSF-STAT5 signaling pathways in human PBMCs,

which revealed that upadacitinib blocked all three pathways with similar efficacy (> 90%) and

potency (IC50 values of ~0.10-0.18 µM), while VVD-118313 fully blocked IFNα-mediated

phosphorylation of STAT1 with an IC50 value of ~0.02 µM, partially blocked IFNγ-mediated

phosphorylation of STAT1 with an Imax of ~50%, and did not inhibit GM-CSF-mediated

phosphorylation of STAT5 (Fig. 5g and Extended Data Fig. 8b).

To further explore the distinct pharmacological profile of VVD-118313, we compared the

compound to other JAK inhibitors in a panel of cytokine-induced STAT phosphorylation assays

in human PBMCs. Consistent with our other results (Fig. 4a-d), VVD-118313, at both test

concentrations (0.1 and 1 µM), produced a near complete blockade of JAK1-dependent IFNα-

STAT1 and IL-6-STAT3 signaling and a partial inhibition of IL-2-STAT5 signaling, while showing

negligible effects on JAK2-dependent GM-CSF-STAT5 signaling (Fig. 5h). This profile was

differentiated from the other tested JAK inhibitors – tofacitinib, upadacitinib or itacitinib – all of

which showed weaker potency than VVD-118313 in the IFNα-STAT1 and IL-6-STAT3 signaling

assays, but much greater activity in the IL-2-STAT5 and GM-CSF-STAT5 assays (Fig. 5h and

Extended Data Fig. 8c). The allosteric TYK2 inhibitor BMS-986165 displayed its greatest

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

potency, as expected, in suppressing IFNα-STAT1 signaling, followed by IL-6-STAT3 and IL-2-

STAT5 signaling, while being inactive against GM-CSF-STAT5 signaling (Fig. 5h and Extended

Data Fig. 8c). Finally, we attempted to measure the effects of VVD-118313 and other JAK

inhibitors on JAK1 phosphorylation, but the signals were too low to visualize in human PBMCs.

Taken together, our studies in primary immune cells illuminate a unique pharmacological

profile of allosteric covalent ligands engaging JAK1_C817 compared to other JAK inhibitors that

includes: i) the strong blockade of cytokine pathways, like IFNα-STAT1 and IL-6-STAT3

signaling, that depend on the catalytic functions of JAK1; ii) the partial inhibition of cytokine

pathways that depend on the scaffolding functions of JAK1 (IFNγ-STAT1) or the activity of

multiple JAK subtypes (IL-2-STAT536); and iii) the sparing of cytokine pathways that depend

predominantly on the activity of other JAK subtypes (GM-CSF-STAT5 (JAK2), IL-12-STAT4

(TYK2)).

Discussion

Despite the potential benefits afforded by allosteric over orthosteric kinase inhibitors, which

include not only improvements in selectivity due to interactions with less conserved pockets, but

also avoidance of direct ATP competition for binding, the identification of ligandable and

functional allosteric sites remains challenging56–58. Allostery is often context-dependent and,

therefore, may not be detected in more conventional high-throughput assays with purified

kinases and simple peptide substrates, especially if these assays only use truncated catalytic

domains59. Existing allosteric kinase inhibitors have largely been discovered serendipitously or

with detailed knowledge of endogenous regulatory mechanisms56–58. Examples include the “type

III” inhibitors of MEK1/260, LIMK61, and AKT62, which bind to a pocket adjacent to the ATP-

binding site, and the “type IV” inhibitors of Bcr-Abl45,46,63,64, MAPKs65, and receptor tyrosine

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

kinases66,67, which bind to allosteric sites distal to the ATP-binding pocket. We have shown here

that chemical proteomics offers a distinct way to discover allosteric inhibitors of kinases.

The profiling of simple electrophilic fragments provided initial evidence of covalent

ligandability of a cysteine that is found in the JH2 pseudokinase domain of JAK1 (C817) and

TYK2 (C838), but not in JAK2 or JAK3. This insight was then efficiently progressed to inhibitory

chemical probes by the coordinated use of targeted chemical proteomic and cell-based

functional assays, furnishing an advanced compound VVD-118313 that site-specifically

engages and inhibits JAK1 in primary immune cells with low-nanomolar potency, while showing

negligible effects on JAK2-dependent signaling pathways. Despite also engaging TYK2_C838

at a concentration < 1 µM, VVD-118313 had a more subtle functional impact on this kinase, as

we did not observe inhibition of TYK2-dependent cytokine signaling by this compound in human

immune cells. These data indicate that VVD-118313 acts as a functionally selective JAK1

inhibitor and overcomes the long-standing challenge confronted by orthosteric inhibitors of

targeting JAK1 while sparing JAK2. As JAK3 only forms heterodimers with JAK1, we did not

assess whether VVD-118313 independently inhibits JAK3 activity in cells, but we do not

anticipate cross-reactivity with JAK3 because it lacks the corresponding liganded cysteine.

Our initial mechanistic studies indicate that VVD-118313 may inhibit JAK1 by blocking

trans-phosphorylation of the activation loop of this kinase. This effect was much stronger for

VVD-118313 compared to orthosteric JAK inhibitors, and we even observed some attenuation

of JAK1 transphosphorylation for the C817A mutant. Our data thus point to a strong potential for

allosteric regulation of JAK1 phosphorylation by the VVD-118313-binding pocket. Considering

this pocket mirrors the myristate-binding pocket of ABL45–47, it is tempting to speculate that

endogenous metabolites might also bind to JAK1 at this site to regulate its function. We also

wonder how many additional kinases may possess this ligandable pocket and prove amenable

to a similar mode of allosteric small-molecule regulation. In this regard, structure-based

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

alignment of 497 human kinase domains reveals a diversity of cysteines at various positions

proximal to the Bcr-Abl myristate pocket68.

The remarkable proteome-wide selectivity displayed by VVD-118313 for JAK_C817

across more than 14,000 quantified cysteines in human and mouse immune cell proteomes

supports the broader utility of this compound as a cellular probe to investigate the specific

biological functions of JAK1. Indeed, using VVD-118313, we discovered that JAK1 makes

differential contributions to IL-6-STAT3 signaling in human PBMCs versus mouse splenocytes,

a finding that may have been obscured in past experiments with other JAK1 inhibitors due to

their lack of subtype selectivity. We also found that the TYK2 inhibitor BMS-986165 was

noticeably more potent in blocking IL-6-STAT3 signaling in mouse splenocytes compared to

human PBMCs (Extended Data Fig. 6f and Fig. 5h). Thus, by using a combination of allosteric

inhibitors with high subtype selectivity, we have provided evidence for species and/or immune

cell type differences in the relative contributions of JAK family members to an important cytokine

signaling pathway. Curiously, splenocytes from TYK2–/– mice have been reported to have

unperturbed IL-6-STAT3 signaling69,70, which could indicate that, in this setting, JAK1

compensates for chronic TYK2 disruption. VVD-118313 should also help to illuminate JAK1

contributions to other signaling pathways, including, for instance, PI3K/AKT, and

MAPK/ERK/p38 and p38 kinase signaling71–73.

Projecting forward, we believe that, while VVD-118313 was capable of inhibiting JAK1 in

mice, the full utility of this chemical probe for in vivo studies would benefit from improvements in

its pharmacokinetic properties. We also wonder if further exploration of the SAR might uncover

compounds that show greater functional activity for TYK2, which could provide an additional

class of useful chemical probes that act as dual allosteric JAK1/TYK2 inhibitors. From a

translational perspective, it is enticing to consider the possibility that covalent allosteric JAK1

inhibitors may circumvent some of the systemic toxicities associated with pan-JAK inhibition in

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

humans. Alternative ways to address this problem have been brought forward, including tissue-

restricted JAK inhibitors, such as gut-restricted izencitinib (TD-1473) for ulcerative colitis74, and

lung-restricted nezulcitinib (TD-0903) for COVID-1975; however, these compounds have not yet

displayed efficacy in clinical studies in humans76,77, pointing to the potential need for systemic

exposure. Of course, it is possible that selective allosteric JAK1 inhibitors may also sacrifice a

proportion of the efficacy observed with systemic pan-JAK inhibitors, but we believe our cellular

studies showing that VVD-118313 matches the activity of pan-JAK inhibitors in suppressing

multiple human cytokine pathways (e.g., IFNa, IL-6) should encourage further pursuit of highly

selective JAK1 inhibitors for immunological disorders.

Finally, we believe that our findings provide another compelling example of the utility of

chemical proteomics for the discovery of small molecules that act by unconventional

mechanisms41,78–82. Chemical proteomic platforms like ABPP have a distinct advantage of

evaluating compounds against thousands of sites on endogenously expressed proteins and can

thus uncover ligandable pockets that may be missed by more conventional assays performed

with purified proteins or protein domains. Nonetheless, chemical proteomics is still principally a

binding assay and interpreting how newly discovered small-molecule interactions affect the

functions of proteins can be technically challenging. Here, we benefited from the availability of

robust cell-based activity assays for JAK1 and, in particular, structural information that

emphasized the potential functionality of a conserved pocket adjacent to the covalently liganded

C817 residue47. As the structures of more full-length proteins are solved or accurately

predicted83,84, the integration of this information with global small-molecule interaction maps

furnished by chemical proteomics should facilitate the discovery of additional cryptic functional

and druggable allosteric pockets on a broad range of proteins.

Acknowledgements

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

This work was supported by the N.I.H. (R35 CA231991) and a Sir Henry Wellcome Postdoctoral

Fellowship (210890/Z/18/Z) awarded to (M.E.K). We thank K. Yao, P. Gao, F. Zhang, X. Li, W.

Wu, X. Jia and M. Xu for their contribution to the synthetic chemistry and B. Melillo for guidance

with chemical analysis.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
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available under aCC-BY-NC-ND 4.0 International license.

Table 1. Engagement and inhibitory activity of covalent ligands targeting JAK1_C817.

Engagement (TE50, μM, 1 h, in vitro) for JAK1_C817 or TYK2_C838 determined by targeted
TMT-ABPP in human cell lysates. Data are mean values ± S.D. from two-three independent
experiments with the exception of values marked with †, which were from a single experiment.
JAK1 inhibition (IC50) determined using HTRF assays measuring IFNα (100 ng/mL, 30 min)-
stimulated STAT1 phosphorylation or IL-6 (25 ng/mL, 30 min)-stimulated STAT3
phosphorylation in human PBMCs pretreated with compounds for 2 h. Compounds were tested
as single stereoisomers except where noted§. Data are mean values ± S.D. from two
independent experiments except where noted (‡n=3, †n=1). ND – not determined. NA – not
applicable for a non-covalent orthosteric inhibitor.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Figure 1. Discovery of a ligandable cysteine in the JAK1/TYK2 pseudokinase domain. a,

Competition ratios of IA-DTB-labeled and enriched cysteine-containing peptides quantified in
MS-ABPP experiments performed using proteomes from human T cells treated in situ with the
cysteine-reactive small-molecule fragments KB02 and KB05 (50 µM, 1 h) or DMSO control. b,
Relative MS3 signal intensity values for all quantified IA-DTB-labeled, cysteine-containing
peptides in JAK1 in KB02- or KB05-treated T cells compared to DMSO-treated T cells. The
KB02- and KB05-liganded cysteine C817 is highlighted in red. Horizontal black bars indicate the
median signal intensity for all other quantified JAK1 cysteines. For a and b, data are mean
values combined from soluble and particulate proteomic analyses from two independent
experiments performed previously41. c, Domain structure of JAK1. C817 and select gain- (red)
or loss- (green) of-function mutations in the pseudokinase (JH2) domain are highlighted. d,
Partial amino acid sequence alignment of human JAK family proteins. Electrophilic fragment-
liganded cysteines in JAK1 (C817) and TYK2 (C838) are highlighted in yellow. Red bar
indicates the tryptic peptide containing JAK1_C817. e, Overlay of the x-ray crystal structures of
the JAK1 JH2 domain (PDB 4L00) and ABL kinase domain (PDB 5MO4), highlighting the
proximity of JAK1 C817 (yellow spheres) to the ABL myristate-binding pocket (pink). The
allosteric ABL inhibitor asciminib (green) and orthosteric inhibitor nilotinib (orange) are show in
stick representations. f, Structure of compound 1a (*single stereoisomer, absolute configuration
not assigned). g, Concentration-dependent engagement of JAK1_C817 by 1a determined by
targeted MS-ABPP experiments (1 h compound treatment of human PBMC or Jurkat cell
proteome). TE, target engagement. h, Concentration-dependent inhibition of IFNα-stimulated
STAT1 phosphorylation (pSTAT1) by 1a in human PBMCs. Cells were treated with 1a for 2 h
followed by 100 ng/mL of IFNα for 30 min, lysed, and pSTAT1 signals measured by HTRF. Data
for g, h are mean values ± S.D. from n = 3 (g) or n = 2 (h) independent experiments.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Figure 2. Optimization of covalent allosteric JAK1 inhibitors. a, Structures of VVD-118313

(compound 5a), stereoisomers (5b-d), and key precursors (2a, 3a, and 4). Compounds 2a, 3a,
and 5a-d were tested as single stereoisomers. Absolute configuration is shown where known. b,
Correlation between engagement of JAK1_C817 (pTE50) determined for compounds as
described in Fig. 1g and inhibition of cytokine-induced STAT1/3 phosphorylation
(pSTAT1/pSTAT3; HTRF pIC50) determined for compounds tested as described in Fig. 1h and
Table 1. R2 values comparing pTE50 values to pIC50 values for pSTAT1 (black circles) and
pSTAT3 (gray squares) were determined by linear regression. c, pIC50 values for inhibition of
IFNα-stimulated STAT1 and IL-6-stimulated STAT3 phosphorylation for representative
compounds determined as described in Fig. 1h and Table 1. For b, c, Data are mean –log-
transformed values from two independent experiments. d, Global cysteine reactivity profile for
VVD-118313 (5a) (1 µM, 3 h, in situ) in primary human PBMCs. Data represent mean ratio
values (DMSO/VVD-118313) for IA-DTB-labeled, cysteine-containing peptides quantified from
two replicate cell treatment experiments analyzed in a single MS-ABPP experiment. Ratio
values for JAK1_C817 (red) and TYK2_C838 (blue) are highlighted. Quantified cysteines with
ratios ≥ 4 (≥ 75% engagement) are marked. e, Concentration-dependent reactivity profiles for
cysteines engaged by VVD-118313 in human PBMCs (0.01-10 μM, 3 h, in situ). Data are mean
values from VVD-118313-treated cells shown as a percentage of DMSO-treated cells from two
replicate cell treatment experiments analyzed in a single MS-ABPP experiment. f, Left, structure
of alkyne probe 6. Right, gel-ABPP experiment showing labeling of recombinant WT-JAK1 and
C810A-JAK1, but not C817A-JAK1, expressed in 22Rv1 cells with alkyne probe 6 (0.1 μM, 2 h,
in situ). The labeling of WT-JAK1 is blocked by pretreatment with VVD-118313 (0.01-1 μM, 2 h,
in situ). Below, western blot showing JAK1 expression in gel-ABPP experiment. Data are from a
single experiment representative of two independent experiments.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Figure 3. VVD-118313 inhibits JAK1 through engagement of C817. a, Representative

cytokine signaling pathways involving different JAK family members. *JAK1 serves a scaffolding
role for IFNg-STAT1 signaling31,38,54. b, Western blots showing effects of VVD-118313 (5a) and
the pan-JAK inhibitor tofacitinib (Tofa) on JAK1 phosphorylation (pJAK1; Y1034/Y1035
phosphorylation detected with (D7N4Z) Rabbit mAb #74129, CST) and IFNα-stimulated STAT1
(JAK1-dependent), IL-6-stimulated STAT3 (JAK1-dependent), and prolactin (PRL)-stimulated
STAT5 (JAK2-dependent) phosphorylation in 22Rv1 cells expressing WT-, C810A-, or C817A-
JAK1. Cells were treated with compounds (2 µM) for 2 h and then stimulated with IFNα (100

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

ng/mL, 30 min), IL-6 (50 ng/mL, 30 min) or PRL (15 ng/mL, 15 min) prior to analysis. c, d,
Quantification of pSTAT1/3/5 (c) and pJAK1 (d) signals from (b). Signal intensities were
normalized relative to the unstimulated WT-JAK1-transfected control for each experiment. Data
are mean values ± S.E.M. from three independent experiments. Significance was determined by
two-way ANOVA with Dunnett’s post-hoc test and reported relative to stimulated, DMSO-treated
control of respective JAK1 construct. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. e,
Concentration-dependent effects of VVD-118313 (5a) on IFNα-stimulated pSTAT1 (left), IL-6-
stimulated pSTAT3 (middle), and pJAK1 (integrated from both IFNα- and IL-6-stimulations) in
22Rv1 cells expressing WT-JAK1. Data are mean values ± SEM from two (pSTAT1, pSTAT3)
or three (pJAK1) independent experiments. See Extended Data Fig. 3b, c for corresponding
western blots. f, Concentration-dependent effects of VVD-118313 (5a; 0.01 – 5 µM, 2 h) and
BMS- 986165 (BMS, 1 or 5 µM, 2 h) on TYK2 phosphorylation (pTYK2) and IFNα-stimulated
STAT1 phosphorylation in 22Rv1 cells expressing recombinant WT-TYK2 or a C838A-TYK2
mutant. Left, representative western blots. Middle and right, quantification of pSTAT1 (middle)
and pTYK2 (right) signals normalized to unstimulated control cells expressing WT-TYK or
C838A-TYK2. Data are mean values ± S.E.M from three independent experiments. Significance
was determined by two-way ANOVA with Dunnett’s post-hoc test. P-values are only shown for
the lowest concentration of each compound that displayed significance for inhibition of pSTAT1
and pTYK2. ****P<0.0001, ***P<0.001.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Figure 4. VVD-118313 selectively inhibits JAK1 signaling in primary human immune cells
and mice. a-d, Effects of VVD-118313 (5a), stereoisomeric mixture 5, and tofacitinib (Tofa) on
JAK-STAT signaling pathways in human PBMCs. PBMCs were treated with compounds at the
indicated concentrations for 2 h prior to stimulation with IFNα (a; 100 ng/mL, 30 min), IL-6 (b; 25
ng/mL, 30 min), IL-2 (c; 20 U/mL, 15 min), or GM-CSF (d; 0.5 mg/mL, 15 min). Upper,
representative western blots. Lower, Quantification of pSTAT signal intensities shown as a
percent of the stimulated DMSO-treated control cells for each assay. Data are mean values ±
S.E.M. from three (IL-6, IL-2) or four (IFNα, GM-CSF) independent experiments. Significance
determined by one-way-ANOVA with Dunnett’s post-hoc test. P-values are only shown for the
lowest concentration of each compound that displayed significance for inhibition of pSTAT. All
higher concentrations were similarly statistically significant. ****P<0.0001, ***P<0.001, **P<0.01,
*P<0.05. e, Effects of VVD-118313 (5a), compound 5, BMS-986195 (BMS), and tofacitinib
(Tofa) on IL-12-stimulated STAT4 phosphorylation in phytohemagglutinin (PHA-P)/IL-2-
generated PBMC-derived T-blasts. Cells were treated with compounds at the indicated
concentrations for 2 h prior to stimulation with IL-12 (12.5 ng/mL, 15 min). Data are mean values

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

± S.E.M. normalized to DMSO controls from three independent experiments and significance
was determined as for (a-d). f, g, Reactivity profiles for JAK1_C816 (f) and all quantified JAK1
cysteines (g) from proteomic lysates of spleen tissue from mice treated with vehicle (dose 0
mg/kg) or compound 5 (25 or 50 mg/kg, s.c. 2 x 4 h). Data are mean values ± S.D. from
compound 5-treated mice shown as a percentage of vehicle-treated mice (n = 4 animals/group
analyzed in a single targeted MS-ABPP). In g, bars represent median reactivity values for all
JAK1 cysteines other than C816. Significance determined by one-way ANOVA with Dunnett’s
post hoc test. ****P<0.0001. h, Ex vivo cytokine stimulation of splenocytes from mice treated
with vehicle or 5 (25 mg/kg, s.c., 2 x 4 h); IFNα (100 ng/mL, 30 min), IL-2 (20 U/mL, 15 min), IL-
6 (10 ng/mL, 30 min) or GM-CSF (10 ng/mL, 15 min). Data are mean values ± S.E.M., from
three (IFNα, IL-2) or one (IL-6, GM-CSF) independent experiments, each containing n = 3 mice
per treatment group. Significance determined by two-way ANOVA with Šidák’s post hoc test.
****P<0.0001.

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

Figure 5. Mechanistic properties and distinct activity profile of allosteric JAK1 inhibitors.
a, Substrate assay quantifying ATP turnover by recombinant purified JAK1 (residues 438-1154
fused to GST) treated with DMSO, VVD-118313 (5a) or tofacitinib (Tofa) (0.001 - μM, 30 min)
prior to addition of an IRS-1 peptide substrate (0.2 µg/mL) and ATP (50 µM, 1 h). Data are
mean values ± S.D. from two independent experiments. b, Western blots measuring JAK1
phosphorylation (pJAK1) from anti-HA immunoprecipitations (IPs) of HA-tagged kinase dead
(K908E) JAK1 (WT or C817A mutant) expressed in 22Rv1 cells alongside catalytically active
FLAG-tagged JAK1 (WT or C817A mutant). c, d, Quantification of pJAK1 signals from anti-HA-
IPs shown in b. Panel c shows pJAK1 signals from anti-HA IPs from DMSO-treated cells co-
expressing the indicated combinations of K908E-JAK1-HA or C817A/K908E-JAK1-HA with WT-

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 bioRxiv preprint doi: https://doi.org/10.1101/2022.01.31.478302; this version posted February 1, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

JAK1-FLAG or C817A-JAK1-FLAG. Panel d shows pJAK1 signals for the same anti-HA IPs
from VVD-118313 (5a; 2 µM, 2 h)-treated 22Rv1 cells. Data in c are normalized to signals in
22Rv1 cells expressing K908E-JAK1-HA and WT-JAK1-FLAG. Data in d are normalized to
signals in DMSO-treated control cells Data for c and d are mean values ± S.E.M. from three
independent experiments. Significance was determined by two-way ANOVA with Dunnett’s
post-hoc test. ***P<0.001, ****P<0.0001. e, Upper, western blots showing that both K908E- and
K908E/C817A-JAK1 mutants support IFNγ-stimulated (50 ng/mL, 30 min), but not IFNα-
stimulated (100 ng/mL, 30 min) STAT1 phosphorylation (pSTAT1) in 22Rv1 cells. WT-JAK1
supports both cytokine pathways. Lower, quantification of western data. Data are mean values ±
S.E.M. from three independent experiments. pSTAT1 signals were normalized to the maximum
signal, which was generated by IFNγ-stimulated WT-JAK1 transfected cells. Significance was
determined by two-way ANOVA with Tukey’s post-hoc test. ****P<0.0001, ***P<0.001. ns – no
significance difference between IFNα-stimulated K908E-JAK1-HA- or K908E/C817A-JAK1-HA-
expressing cells and mock cells. f, Left, western blots showing the effects of VVD-118313 (5a;
0.1-5 µM, 2 h) and tofacitinib (Tofa; 1 µM, 2 h) on IFNγ-dependent STAT1 phosphorylation
(pSTAT1) in 22Rv1 cells expressing K908E-JAK1-HA or K908E/C817A-JAK1-HA. Right,
quantification of western data. Data are mean values ± S.E.M. from three independent
experiments. Significance determined by two-way ANOVA with Dunnett’s post-hoc test.
*P<0.05, **P<0.01, ****P<0.0001, ns – non-significant. g, Concentration-dependent effects of
VVD-118313 (5a) or upadacitinib (Upa) on IFNα-dependent STAT1, IFNγ-dependent STAT1,
and GM-CSF-dependent STAT5 phosphorylation in human PBMCs. pSTAT signals were
normalized to the DMSO-treated cytokine-stimulated control in each assay. Dose-response
curves are mean values ± S.D. of two biological replicates used to estimate IC50 values by
fitting data to a 4PL model. h, Left, western blots comparing the effects of VVD-118313 (5a) and
a panel of JAK inhibitors on the indicated cytokine-stimulated pSTAT pathways in human
PBMCs. Right, quantification of western data. Data are mean values ± S.E.M. from two (IL-6) or
three (IFNα, IL-2, GM-CSF) independent experiments. Significance determined by one-way
ANOVA with Šidák’s post-hoc test. *P<0.05, ****P<0.0001, ns – non-significant

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