ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Covalent Stapling of the Cereblon Sensor Loop Histidine Using Sulfur-Heterocycle Exchange.

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO2Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting.

A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors.

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Mitochondrial pyruvate supports lymphoma proliferation by fueling a glutamate pyruvate transaminase 2-dependent glutaminolysis pathway.

The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)-like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.

Cereblon covalent modulation through structure-based design of histidine targeting chemical probes.

Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

Small molecule inhibition of deubiquitinating enzyme JOSD1 as a novel targeted therapy for leukemias with mutant JAK2.

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase.

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors.

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma.

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.

A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR-/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma.

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.

Discovery of Covalent MKK4/7 Dual Inhibitor.

MKK4/7 are kinases that phosphorylate JNKs and regulate the MAPK signaling pathway. Their overexpression has been associated with tumorigenesis and aggressiveness in cancers such as breast, prostate, non-small cell lung, and pediatric leukemia, making them a potential target for inhibitor development. Here, we report the discovery, development, and validation of a dual MKK4/7 inhibitor, BSJ-04-122, that covalently targets a conserved cysteine located before the DFG motif and displays excellent kinome selectivity. BSJ-04-122 exhibits potent cellular target engagement and induces robust target-specific downstream effects. The combination of the dual MKK4/7 inhibitor with a selective, covalent JNK inhibitor demonstrated an enhanced antiproliferative activity against triple-negative breast cancer cells. Taken together, the results show that BSJ-04-122 represents a pharmacological probe for MKK4/7 and credential covalent targeting as a way to explore the therapeutic potential of these kinases.