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. 2022 Oct 24;13(11):1819–1826. doi: 10.1021/acsmedchemlett.2c00414

Covalent Modification of the JH2 Domain of Janus Kinase 2

Sean P Henry †, Maria-Elena Liosi †, Joseph A Ippolito †, Fabian Menges †, Ana S Newton †, Joseph Schlessinger ‡, William L Jorgensen †,*
PMCID: PMC9661697  PMID: 36385940

Abstract

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Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30-fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.

Keywords: JAK2 kinase, pseudokinase domain, covalent ligands, kinase inhibitors JH2/JH1 selectivity

Janus kinases (JAKs) are a unique class of non-receptor tyrosine kinases that possess two ATP binding domains. The active JH1 kinase domain phosphorylates signal transducer and activator of transcription (STAT) proteins,1,2 and the pseudokinase (JH2) domain regulates the JH1 domain’s intrinsic kinase activity.3−5 There are four JAKs (JAK1, JAK2, JAK3, and TYK2), and dysregulating mutations in these kinases lead to various forms of leukemia, lymphoma, myeloproliferative neoplasms, and primary immunodeficiencies.2 A host of clinically approved drugs target the JH1 domains of JAKs,6−8 and the TYK2 JH2 ligand deucravacitinib was just approved for moderate-to-severe plaque psoriasis.9

To validate JH2 domains as therapeutic targets, JH2/JH1 selectivity is required. For TYK2 JH2, deucravacitinib’s selectivity is achieved by a deuteromethylamide motif near the hinge region.10 In the non-covalent JAK2 JH2 ligands developed in our lab,11−17 selectivity is promoted through a combination of a carboxylate that interacts with Thr555 and Arg71513 and a terminal aryl pharmacophore that interacts with Arg715 and Trp718.12,14 Acquiring selectivity is challenging for kinase inhibitors, owing to the abundance of kinases and the sequence conservation in their ATP-binding sites. Thus, the use of targeted covalent inhibitors (TCIs) has been receiving increased attention as an alternative approach to enable high kinase selectivity.18−21 The Janus kinases are no exception, and covalent inhibitors acting on the JAK3 JH1 domain have been developed.22−30 In addition, one compound, xanthatin, has been shown to bind covalently to Cys243 of the FERM domain of JAK2.31

Analysis of the available crystal structures of three JH2 domains (JAK1, JAK2, and TYK2) indicates the presence of a cysteine in the catalytic loop, in contrast to the four JH1 domains (JAK1, JAK2, JAK3, TYK2) that each possess an alanine at this position (Figure 1A, red arrow).15,30,32−35 These cysteines are in a different location than the previously targeted cysteine in JAK3 JH1, which is uniquely located in the front pocket (Figure 1A, gold arrow). We began by modeling complexes with the ligand JNJ7706621 (1) as a reference (Figure 1B), which indicated that the cysteines in the catalytic loop of the JH2 domains should be targetable. Though no crystal structure of JAK3 JH2 has been reported yet, a UniProt sequence alignment was performed to compare the four JAK JH2 domains and determine if either of these targetable cysteines is present in JAK3 JH2.36 This alignment shows that the cysteine in the catalytic loop of the other JH2 domains is replaced with a serine in JAK3 JH2 (Figure 1C, red box). Furthermore, the front pocket cysteine found in the JAK3 JH1 domain is replaced with an alanine in JAK3 JH2 (Figure 1C, gold box). A summary of these targetable cysteines as well as residues in the equivalent position in each domain is shown in Figure 1D.

Figure 1.

Figure 1

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(a) Targetable cysteines within the JH1 and JH2 domains of the JAK kinases. The cysteines in the JH2 domains of JAK1, JAK2, and TYK2 are in the catalytic loop (red arrow). The JH1 domains of all four JAKs possess alanines in this position. JAK3 JH1 possesses a unique cysteine in the front pocket (gold arrow) that is replaced with alanines, serines, and prolines in the other domains. The crystal structures of JAK1 JH1 (PDB: 4EI4), JAK1 JH2 (PDB: 4L00), JAK2 JH1 (PDB: 5USY), JAK2 JH2 (PDB: 5USZ), JAK3 JH1 (PDB: 5TTS), TYK2 JH1 (PDB: 4GJ2), and TYK2 JH2 (PDB: 7K7Q) are used to show these residues. JAK2 JH2 (PDB: 5USZ) bound to compound 1 is used as a template. (b) Structure of compound 1. (c) Sequence alignment of human JAK1, JAK2, JAK3, and TYK2 focusing on the catalytic loop and front pocket of the JH2 domain. The cysteines in the catalytic loop are replaced with a serine in JAK3 JH2 (red box), and the front pocket residues (gold box) show an alanine in JAK3 JH2. Conserved residues across all four domains are shown to indicate alignment (green boxes). The UniProt IDs for each domain are P23458 (JAK1), O60674 (JAK2), P52333 (JAK3), and P29597 (TYK2).36 (d) Identity of each front pocket and catalytic loop residue in all JAK JH1 and JH2 domains. Cysteines in the catalytic loop are red, and cysteines in the front pocket are gold.

Given the exploitable cysteines in the ATP pocket of the JH2 domain, and their absence in the associated JH1 domains, evaluation of their targetability to enhance JH2 selectivity was pursued. JNJ7706621 (1) was selected for derivatization, due in part to its use as a control in our prior studies and its development into a tracer.151 was also found to bind the JAK2 JH2 and JH1 domains with nearly equal affinity,16 allowing us to probe the variation in selectivity induced by covalent modification. Crystal structures and modeling of 1 bound to both the JAK2 JH2 and JH1 domains (Figure 2) indicated that Cys675 in the JH2 domain should be accessible via substituents placed at the 3- or 4-position of the 2,6-difluorobenzamide motif.17 In the corresponding JH1 ATP-site, this cysteine is replaced by alanine, and access to it is blocked by Arg980. Arg980 is held in place by a charge–charge interaction with Asp976, closing the pocket. In JAK2 JH2, Asp976 is converted into Asn673 and Arg980 is converted into Lys677. This results in a disrupted charge–charge interaction that would otherwise exist with the side chain of Lys677 and allows access to Cys675 in JH2.

Figure 2.

Figure 2

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Crystal structures of 1 bound to (a) WT JAK2 JH2 (PDB: 5USZ)17 and (b) WT JAK2 JH1 (PDB: 5USY).17 Cys675 is accessible in the JH2 domain (blue arrow) in an open pocket, while in JH1 it is replaced by Ala978 and the pocket is closed (red arrow).

To consider viable linker–warhead constructs for attachment to 1, de novo design was employed using the molecule growing program BOMB.37 Acrylamides were selected as the warhead, given their precedence in covalent drugs,38 their established in vitro and in vivo utility,39,40 the ability to fine-tune their reactivity with substituents on the α- or β-positions,41,42 and our prior success with them in covalent modification of Cys181 in a variant of HIV-1 reverse transcriptase.43 Among the constructs that were modeled, 3-methylene and 3-ethylene acrylamides seemed particularly promising (Figure 3). These appendages are predicted to allow placement of the β-position of the acrylamide at distances of 3.80 and 3.48 Å, respectively, to the cysteine sulfur atom in low-energy conformations with little change in the positioning of the triazole core in the hinge region. The predicted structure with the ethylene linker also features a hydrogen bond between the acrylamide nitrogen and the side-chain carbonyl oxygen of Asn678. The flexibility and length of this linker should permit repositioning of the acrylamide as needed without inducing undue steric effects or strain in the non-covalent and covalent complexes.

Figure 3.

Figure 3

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Modeled derivatives of 1 with (A) a 3-methylene acrylamide and (B) a 3-ethylene acrylamide group bound to JAK2 JH2. Complexes were generated with BOMB starting from the crystal structure for JAK2 JH2 with 1 (PDB: 5USZ).

The series of new analogues began by replacing the sulfonamide of 1 with an N-methylamide to improve cell permeability and reduce mass.14 The resultant compound provided a baseline for the subsequent studies. Attachment of the acrylamide warhead was pursued at the 3-position of the difluorophenyl ring with methylene, ethylene, and ethyleneoxy linkers, and at the 4-position with an ethyl linker. In addition, attachment of a β-methyl group to the 3-ethyl acrylamide was carried out to consider reduced reactivity.

The syntheses began with formation of functionalized 2,6-difluorobenzoates (4a–d, Scheme 1). Introduction of the 3-methylene linker began with protection of 2,6-difluoro-3-formylbenzoic acid as a methyl ester using MeI and K2CO3. The methylene amine was introduced in a protected form by attachment of (R)-2-methylpropane-2-sulfinamide mediated by Ti(OEt)4, followed by reduction. This also resulted in transesterification to the ethyl ester, which was subsequently saponified to yield 4a.

Scheme 1. Synthesis of Functionalized Difluorobenzoic Acids.

Scheme 1

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Reagents and conditions: (a) MeI, K2CO3, DMF, rt; (b) 1, I-2-methylpropane-2-sulfinamide, Ti(OEt)4, THF, 65 °C; 2, NaBH4, −78 °C to rt; (c) LiOH·H2O, H2O, EtOH, THF, rt; (d) 2 M BH3·Me2S, THF, 0 °C to rt; (e) potassium 2-(Boc-aminoethyl)trifluoroborate, Pd(OAc)2, RuPhos, Cs2CO3, toluene, H2O, 110 °C; (f) NaIO4, RuCl3·xH2O, MeCN, EtOAc, H2O, 0 °C; (g) SOCl2, MeOH, 0 °C to rt; (h) tert-butyl (2-bromoethyl)carbamate, K2CO3, DMF, rt; (i) 2 M NaOH, THF, MeOH, rt.

The 3- and 4-ethylene linkers were introduced by a palladium-mediated cross-coupling between an aryl bromide and potassium 2-(Boc-aminoethyl)trifluoroborate.44 It is noted that the 2,6-difluorobenzoates are particularly sensitive to this reaction. With a methyl 2,6-difluorobenzoate as the aryl bromide, debromination was observed with Pd(dppf)Cl2·DCM as the catalyst, and no reaction occurred with Pd(OAc)2 and RuPhos. This appeared to be an electronic effect, as 2,6-difluorobenzyl alcohol acting as aryl bromide was a successful Suzuki coupling partner, but only when RuPhos and Pd(OAc)2 were used as ligand and catalyst. Debromination was again observed if Pd(dppf)Cl2·DCM was used with the benzyl alcohols. Following Suzuki coupling to introduce the Boc-protected ethylamines, the benzylic alcohol was then oxidized using NaIO4 and RuCl3·xH2O, yielding the alkylated benzoic acids (4b,c).

A 3-ethyleneoxy linker was introduced by protecting the carboxylic acid of 2,6-difluoro-3-hydroxybenzoic acid using thionyl chloride in methanol (2d). The subsequent phenol was alkylated with tert-butyl (2-bromoethyl)carbamate, and the methyl ester was saponified to yield 4d. The benzoic acids and benzoates synthesized as shown in Scheme 1 were then coupled to 4-((5-amino-1H-1,2,4-triazol-3-yl)amino)-N- methylbenzamide14 using HATU (Scheme 2). The amines were deprotected, and acrylic or crotonic anhydride was used to yield the desired acrylamides, 7a–f.

Scheme 2. Synthesis of Final Compounds.

Scheme 2

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Reagents and conditions: (a) benzoic acids/lithium benzoates 4a–d, HATU, NMM, DMF, rt; (b) conc. HCl, dioxane, rt; (c) acrylic or crotonic anhydride, TEA, dioxane, rt.

Following synthesis, binding affinities (Kd) were evaluated with the JAK2 JH2 and JH1 domains (Table 1) using the previously reported fluorescence polarization (FP) assay.16 For the JH2 domain, Kd’s were determined both at the standard time of 30 min and at 20 h. The negative control is the tracer in the buffer solution, while the positive control includes enough protein such that all tracer is bound, which is the same protein concentration across all other assays. If the protein were denatured, the positive and negative controls would give the same FP values and a Kd could not be measured, while formation of a covalent bond should lead to a decrease in Kd with increasing incubation time owing to the progressive increase in unbound tracer.18 For comparison, the Kd’s of both sulfonamide 1 and the N-methylamide analogue 7a are shown in Table 1. The selectivities for JH2 over JH1 at 30 min and 20 h are also presented.

Table 1. Binding Affinities of Analogues to the JAK2 JH2 Domain and the JAK2 JH1 Domaina.

graphic file with name ml2c00414_0007.jpg

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a

Binding affinities (Kd) using a previously reported fluorescence polarization (FP) assay.15

b

From ref (13). ND = not determined.

Conversion of the sulfonamide 1 to the N-methylamide (7a) results in 3-fold greater affinity to the JH2 domain. Since 7a lacks a warhead, minimal shift in the Kd of 7a is observed upon lengthening the incubation from 0.5 to 20 h. Introduction of the acrylamides did yield shifts in Kd. The 3-methylene analogue (7b) caused a 7-fold loss of binding affinity relative to 7a at 30 min, though a 1.6-fold improvement in affinity was found after 20 h. These data could reflect unconstructive non-covalent interactions between the methyl acrylamide and the JH2 domain that are slowly overcome by some formation of a covalent bond with Cys675.

Extending the linker to ethylene in 7c improved the affinity to 0.556 μM for the JH2 domain. This was amplified 5-fold after 20 h to 0.110 μM, surpassing even the reference non-covalent analogue 7a. The change in selectivity of 7c for JAK2 JH2 over JAK2 JH1 is noteworthy, improving from 2.3-fold to 11.6-fold on going to 20 h. These results are consistent with the formation of a covalent bond between 7c and JAK2 JH2. It may be noted that JAK2 JH2 possesses seven cysteines. From many crystal structures, Cys616 and 618 form a disulfide bond, and Cys723, Cys747, and Cys787 are well-packed and not expected to be accessible to a ligand, while Cys644 is on the surface of the protein.13−18 Only Cys675 is in the ATP-binding site. Covalent attachment of the ligand to any other cysteine should not displace the tracer and affect the fluorescence measurements.

The addition of a methyl group to produce the (E)-acrylamide 7d causes the affinity to remain constant from 30 min to 20 h (∼0.57 μM). These values are consistent with non-covalent binding to JAK2 JH2; lower reactivity of the warhead and steric effects caused by the methyl group are preventing covalent bond formation with Cys675.45 Comparing these values with the 30 min JAK2 JH2 Kd for 7c (0.556 μM) suggests that the affinities of 7c and 7d at 30 min are the result of non-covalent binding, with the acrylamide tails making minimal contact with the protein.

Shifting the ethylene linker of 7c from the 3-position (meta) to the para-position (7e) yields an analogue with a Kd (0.242 μM) at 30 min nearly the same as for 7a. The para-substitution is more likely to orient the substituent into the solvent, resulting in no new positive or negative interactions with the JH2 domain. However, after 20 h, a 3.2× improvement in affinity is observed, yielding the lowest Kd of 0.075 μM. It is expected that, over time, conformers are sampled in which the linker wraps back and contact is made between the warhead and Cys675. The Kd with JAK2 JH1 is akin to its 3-position counterpart (7c), and the selectivity acquired after 20 h for 7e is also similar to that for 7c.

Further extension of the 3-ethylene linker with an oxygen to give 7f resulted in a JAK2 JH2 Kd at 30 min (0.617 μM) close to the value for the ethylene-linker analogue 7c, and a 4× lowering of the Kd at 20 h to 0.154 μM. All results for 7c and 7f are indicative of significant increases in potency and selectivity with increasing time. Overall, the Kd data indicate covalent bond formation with JAK2 JH2 for at least 7c, 7e, and 7f, and possibly 7b.

To complement the above Kd data, each analogue was incubated with JAK2 JH2 at 4 °C for up to 48 h and subjected to high-pressure liquid chromatography electrospray quadrupole time-of-flight HRMS (HPLC-ESI-QToF MS) experiments. The raw data at 24 h were charge deconvoluted such that the calculated M+H for the JAK2 JH2 domain alone is shown for each experiment (Figure 4, green boxes). If a covalent bond forms between the protein and the ligand, an increase in mass of the protein by the mass of the ligand is expected. As such, the charge-deconvoluted M+L+H is also shown (Figure 4, red boxes). The control analogue 7a (Figure 4A) produces only the M+H peak, consistent with no covalent binding. However, in line with the Kd data, each simple acrylamide produced an M+L+H peak in addition to the M+H peak (Figure 4B, C, and F), the exception being 7e (Figure 4E), in which the protein appears to have degraded. Taken in conjunction with the Kd results above, these results strongly support that a covalent bond is formed between analogues 7b, 7c, and 7f to Cys675 of JAK2 JH2. Introduction of a β-methyl group in the acrylamide prevents covalent binding to the protein, as indicated by the lack of an M+L+H peak (Figure 4D), which is again consistent with the Kd data in Table 1.

Figure 4.

Figure 4

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Charge-deconvoluted mass spectra for JAK2 JH2 after 24 h incubation with analogues shown in Table 1. Shown are the results for compounds 7a–f. Green boxes highlight the mass of the JAK2 JH2 domain alone, whereas red boxes highlight the mass of JAK2 JH2 with covalently bound ligand. The results for 7e likely reflect protein degradation.

The covalent targeting of non-conserved cysteines can be a useful strategy for improving the selectivity of kinase ligands. Selectivity for the JH2 domains of JAKs is important to validate these pseudokinases as therapeutic targets, particularly to prevent confounding effects from simultaneous modification of the associated JH1 domains. The cysteines located on the catalytic loop of three of the JH2 domains of JAKs can provide an avenue to this selectivity via a ligand armed with a covalent warhead. Herein, we have reported the successful covalent modification of the JAK2 JH2 domain. The combined fluorescence polarization and mass spectrometry data are readily explained by displacement of the tracer from the ATP binding site and covalent modification of Cys675. The attachment of acrylamide warheads to a non-selective JAK2 JH1/JH2 binding molecule has provided 3–5-fold shifts in Kd values and, depending on the linker, up to 28-fold selectivity for the JH2 domain over the JH1 domain, as compared to 1.5-fold for the parent ligand 1. Future studies can explore the effects of the reported molecules and analogues on JAK kinase activity and the potential for covalent JH2-binding molecules as therapeutic agents.

Acknowledgments

This work was supported by the U.S. National Institutes of Health (GM32136). This research made use of the Chemical and Biophysical Instrumentation Center at Yale University (RRID: SCR_021738) for mass spectrometry data collection.

Glossary

Abbreviations

JH2

pseudokinase domain

JH1

kinase domain

JAK

Janus kinase

TYK2

tyrosine kinase 2

HRMS

high-resolution mass spectrometry

ESI

electrospray ionization

QToF

quadrupole time-of-flight

ATP

adenosine triphosphate

TCI

targeted covalent inhibitor

FERM

4.1 protein, ezrin, radixin, moesin

WT

wild-type

DMF

N,N-dimethylformamide

THF

tetrahydrofuran

RuPhos

2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate

NMM

N-methylmorpholine

TEA

triethylamine

Kd

dissociation constant

FP

fluorescence polarization

All data are available in the main text or Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00414.

  • Details for the synthetic procedures, compound characterization, computations, and assays (PDF)

  • 1H and 13C NMR of final compounds 7a–7f (PDF)

  • SMILES strings for 7a–7f (XLSX)

The authors declare the following competing financial interest(s): Yale University has submitted a preliminary patent application related to the reported findings.

Supplementary Material

ml2c00414_si_001.pdf (706.9KB, pdf)
ml2c00414_si_002.pdf (1.6MB, pdf)
ml2c00414_si_003.xlsx (10.6KB, xlsx)

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ml2c00414_si_001.pdf (706.9KB, pdf)
ml2c00414_si_002.pdf (1.6MB, pdf)
ml2c00414_si_003.xlsx (10.6KB, xlsx)

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