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Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. [ABSTRACT FROM AUTHOR]

The article reports on a study related to highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015 which inhibit the phosphorylation of the only well-characterized substrate. It describes the materials and methodology including antibodies, electrophoresis, immunoblotting, immunoprecipitation and tissue culture. It concludes that WZ4003 and HTH-01-015 can serve as useful chemical probes to describe the biological roles of the NUAK kinases.

Abstract: The function and higher order structure of β-proline oligomers have been relatively unexplored compared to other foldamer classes due in part to synthetic challenges associated with substituted monomers. Here we report a flexible synthesis of di- and trisubstituted isoxazolidine β-proline monomers that enables access to a wide range of densely functionalized analogs suitably protected for solid-phase synthetic protocols. This strategy utilizes chiral isoxazolines as key intermediates and can readily provide single stereoisomers of the target molecules. [Copyright &y& Elsevier]

Simple Summary: Multiple myeloma (MM) is an incurable disease affecting predominantly elderly people (>65 years of age). Despite improvement in the 10-year survival rate for MM patients due to the advent of medications, including immunomodulatory drugs (IMiDs) (such as pomalidomide and lenalidomide) and proteasome inhibitors, coupled with autologous stem cell transplant, more effective and safe therapies are still needed. Bromodomain-containing protein 9 (BRD9) has been shown to be important for the survival of MM cells. We discovered that drugs that block the function of BRD9 increase the effectiveness of IMiDs and can override resistance to these medications. Our findings suggest that the combined use of IMiDs and drugs that target BRD9 could potentially improve clinical outcomes for MM patients. Progress in the treatment of multiple myeloma (MM) has resulted in improvement in the survival rate. However, there is still a need for more efficacious and tolerated therapies. We and others have shown that bromodomain-containing protein 9 (BRD9), a member of the non-canonical SWI/SNF chromatin remodeling complex, plays a role in MM cell survival, and targeting BRD9 selectively blocks MM cell proliferation and synergizes with IMiDs. We found that synergy in vitro is associated with the downregulation of MYC and Ikaros proteins, including IKZF3, and overexpression of IKZF3 or MYC could partially reverse synergy. RNA-seq analysis revealed synergy to be associated with the suppression of pathways associated with MYC and E2F target genes and pathways, including cell cycle, cell division, and DNA replication. Stimulated pathways included cell adhesion and immune and inflammatory response. Importantly, combining IMiD treatment and BRD9 targeting, which leads to the downregulation of MYC protein and upregulation of CRBN protein, was able to override IMiD resistance of cells exposed to iberdomide in long-term culture. Taken together, our results support the notion that combination therapy based on agents targeting BRD9 and IKZF3, two established dependencies in MM, represents a promising novel therapeutic strategy for MM and IMiD-resistant disease. [ABSTRACT FROM AUTHOR]

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies. [ABSTRACT FROM AUTHOR]

This article focuses on a study related to stereochemical promiscuity in artificial transcriptional activators. The prevailing paradigm driving the discovery of small molecules that perturb biological functions requires ligands of high affinity and specificity for a particular macromolecule. Transcriptional activation is an example of a process initiated by a network of lower affinity interactions and is thus an ideal system in which to explore the development of small molecule regulators of macromolecular complexes. Results reveal that the isoxazolidine small molecule ADs mimic the functional profile of natural ADs in that precise positioning of the amphipathic side chains is not a critical determinant for function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIVrelated malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIVrelated malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X. [ABSTRACT FROM AUTHOR]

Since its introduction by Cravatt and colleagues in 1999, activity‐based protein profiling (ABPP) has become widely utilized throughout academia, government, and industry laboratories to study enzymes spanning numerous gene families in a multitude of biological systems. As a variation of ABPP, competitive ABPP provides a powerful approach to characterize the binding behavior of small molecule probes and clinical drugs throughout the functional proteome. The power and flexibility of competitive ABPP are exemplified by a wide range of creative adaptions which increase assay throughput, enable diverse detection schemes, and support the implementation of this approach within a hybrid target‐based screening platform. We review major developments in competitive ABPP through a compare‐contrast format to provide a useful introduction to this enabling technology for scientists in chemical biology and drug discovery. [ABSTRACT FROM AUTHOR]

SUMOylation is a post-translational modification with important roles in normal physiology and whose dysregulation is associated with human diseases. In this issue of Cell Chemical Biology , Li et al. (2019) describe a covalent, allosteric inhibitor of the SUMO E1 enzyme and demonstrate its anti-tumor activity in preclinical models of colorectal cancer. [ABSTRACT FROM AUTHOR]

Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7). [ABSTRACT FROM AUTHOR]

Deubiquitinating enzymes (DUBs) are a largely understudied and untapped resource in the toolkit of protein degradation functionalities. They comprise a large repertoire of enzymes that remove ubiquitin from substrates in a variety of cellular and pathophysiological contexts, and have enormous potential for research and clinical use. It is only within the last 5 years that potent, selective, and well-characterized small-molecule inhibitors of DUBs have been described. These compounds are now being used to study the biological roles of DUBs. Here, we describe downstream applications of small-molecule inhibitors for studying DUBs and provide a framework for future studies. We highlight recent examples of using these inhibitors to confirm and explore the role of these enzymes in both normal and pathological contexts. These studies represent the first steps in the burgeoning field of pharmacological and chemoproteomic studies of DUBs, which will be critical for the continued advancement of DUB field. [Display omitted] Magin et al. review the development of small-molecule inhibitors of deubiquitinating enzymes (DUBs), and their application in studying DUB function and therapeutic potential. Examples of well-validated inhibitors are given and future perspectives for this growing field are discussed. [ABSTRACT FROM AUTHOR]

Henning et al. (2022) report development of a novel class of agents, bivalent deubiquitinase (DUB)-targeting chimeras (DUBTACs), that can selectively stabilize protein targets. These findings encourage further pursuit of targeted protein stabilization as a new paradigm in chemical biology and drug discovery. [ABSTRACT FROM AUTHOR]

Summary: Mutations in two type‐3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3‐positive AML and for KIT D816V‐positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild‐type (wt) FLT3, FLT3‐internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3‐ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c‐CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ≤2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that while the structure in complex with AMP-PNP is in an "αC-out" inactive conformation, those in complex with type I inhibitors assume an active "Asp-Phe-Gly (DFG)-in" and "αC-in" conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a "DFG-out" conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation. [ABSTRACT FROM AUTHOR]

Acquired ibrutinib resistance due to BTKCys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTKCys481 mutations are usually subclonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTKCys481Ser and BTKWT expressing MYD88-mutated Waldenström macroglobulinemia (WM) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cells and observed reactivation of BTK-PLCγ2-ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTKCys481Ser-expressing cells and showed synergistic cytotoxicity with ibrutinib. ERK1/2 reactivation in ibrutinib-treated BTKCys481Ser cells was accompanied by release of many prosurvival and inflammatory cytokines, including interleukin-6 (IL-6) and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib-treated BTKCys481Ser cells could protect BTKWT MYD88-mutated malignant cells, we used a Transwell coculture system and showed that nontransduced BTKWT MYD88-mutated WM or ABC DLBCL cells were rescued from ibrutinib-induced killing when cocultured with BTKCys481Ser but not their BTKWT-expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on nontransduced BTKWT by coculture with BTKCys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTKCys481 mutations. Our findings show that the BTKCys481Ser mutation drives ibrutinib resistance in MYD88-mutated WM and ABC DLBCL cells through reactivation of ERK1/2 and can confer a protective effect on BTKWT cells through a paracrine mechanism. [ABSTRACT FROM AUTHOR]

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/ threonine kinases that play critical roles in initiating innate immune responses against foreign pathogens and other types of dangers through their role in Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and IL-1Rs recruit adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88), to the membrane, which in turn recruit IRAKs via the death domains in these proteins to form the Myddosome complex, leading to IRAK kinase activation. Despite their biological and clinical significance, only the IRAK4 kinase domain structure has been determined among the four IRAK family members. Here, we report the crystal structure of the human IRAK1 kinase domain in complex with a small molecule inhibitor. The structure reveals both similarities and differences between IRAK1 and IRAK4 and is suggestive of approaches to develop IRAK1- or IRAK4-specific inhibitors for potential therapeutic applications. While the IRAK4 kinase domain is capable of homodimerization in the unphosphorylated state, we found that the IRAK1 kinase domain is constitutively monomeric regardless of its phosphorylation state. Additionally, the IRAK1 kinase domain forms heterodimers with the phosphorylated, but not unphosphorylated, IRAK4 kinase domain. Collectively, these data indicate a two-step kinase activation process in which the IRAK4 kinase domain first homodimerizes in the Myddosome, leading to its trans-autophosphorylation and activation. The phosphorylated IRAK4 kinase domain then forms heterodimers with the IRAK1 kinase domain within the Myddosome, leading to its subsequent phosphorylation and activation. [ABSTRACT FROM AUTHOR]

The PTPN11 oncogene encodes the cytoplasmic protein tyrosine phosphatase SHP2, which, through its role in multiple signaling pathways, promotes the progression of hematological malignancies and other cancers. Here, we employ high-throughput screening to discover a lead chemical scaffold, the benzothiazolopyrimidones, that allosterically inhibits this oncogenic phosphatase by simultaneously engaging the C-SH2 and PTP domains. We improved our lead to generate an analogue that better suppresses SHP2 activity in vitro. Suppression of Erk phopsphorylation by the lead compound is also consistent with SHP2 inhibition in AML cells. Our findings provide an alternative starting point for therapeutic intervention and will catalyze investigations into the relationship between SHP2 conformational regulation, activity, and disease progression. [ABSTRACT FROM AUTHOR]

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients. [ABSTRACT FROM AUTHOR]

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307, 520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-)T cells and had an IC50 as low as 8.4 µM and a TC50 of >100 µM when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 µM). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity. [ABSTRACT FROM AUTHOR]

Wedeveloped a pharmacophore model for type II inhibitors thatwas used to guide the construction of a library of kinase inhibitors.Kinome-wide selectivity profiling of the library resulted in the identificationof a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity againsttwo mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2,as well as pharmacologically well interrogated kinases such as p38α(MAPK14) and ABL. Further investigation of the structure–activityrelationship (SAR) resulted in the identification of potent dual TAK1and MAP4K2 inhibitors such as 1(NG25) and 2as well as MAP4K2 selective inhibitors such as 16and 17. Some of these inhibitors possess good pharmacokineticproperties that will enable their use in pharmacological studies invivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1confirms that the activation loop of TAK1 assumes the DFG-outconformation characteristic of type II inhibitors. [ABSTRACT FROM AUTHOR]

Myeloid differentiation factor 88 (MYD88) L265P somatic mutation is highly prevalent in Waldenström macroglobulinemia (WM) and supports malignant growth through nuclear factor κB (NF-κB). The signaling cascade(s) by which MYD88 L265P promotes NF-κB activation in WM remain unclear. By lentiviral knockdown or use of a MYD88 inhibitor, decreased phosphorylation of the NF-κB gatekeeper IκBα and survival occurred in MYD88 L265P-expressing WM cells. Conversely, WM cells engineered to overexpress MYD88 L265P showed enhanced survival. Coimmunoprecipitation studies identified Bruton tyrosine kinase (BTK) complexed to MYD88 in L265P-expressing WM cells, with preferential binding of MYD88 to phosphorylated BTK (pBTK). Increased pBTK was also observed in WM cells transduced to overexpress L265P vs wild-type MYD88. Importantly, MYD88 binding to BTK was abrogated following treatment of MYD88 L265P-expressing cells with a BTK kinase inhibitor. Inhibition of BTK or interleukin-1 receptor-associated kinase 1 and 4 (IRAK-1 and -4) kinase activity induced apoptosis of WM cells, and their combination resulted in more robust inhibition of NF-κB signaling and synergistic WM cell killing. The results establish BTK as a downstream target of MYD88 L265P signaling, and provide a framework for the study of BTK inhibitors alone, and in combination with IRAK inhibitors for the treatment of WM. [ABSTRACT FROM AUTHOR]

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes. [Display omitted] • High-throughput screen of ∼50k compound library against 8 deubiquitinases • Identification and validation of selective inhibitors for individual DUBs • Study demonstrates importance of validation experiments as part of hit triage • Generation of robust dataset of compound activities against selected DUBs Deubiquitinases (DUBs) have emerged as therapeutically interesting targets due to their involvement in protein turnover in cells. Varca et al. describe a multi-DUB high-throughput screen and orthogonal validation campaign to identify new tool compounds for elucidating DUB function and provide a roadmap for future DUB inhibitor screens. [ABSTRACT FROM AUTHOR]

Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To date, the majority of clinical and preclinical kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential “kinase cysteinome” and discuss strategies for the efficient development of new covalent inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria. [Copyright &y& Elsevier]

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents. • Commonly used DUB inhibitors show a wide range of selectivity and cellular activity • Cellular proteomics of USP7, USP47, USP25, USP28, USP30, UCHL1, and USP1 inhibitors • Use of multiple USP7 inhibitors to confirm known and new targets of USP7 • Practical insights for profiling DUB inhibitors earlier in the development pipeline Bushman et al. profile commonly used inhibitors of deubiquitinating enzymes to assess their biochemical selectivity and cellular activity. USP7 is used to demonstrate proof-of-concept for using potent and selective inhibitors of deubiquitinating enzymes to identify their substrates by mass spectrometry. [ABSTRACT FROM AUTHOR]

The outbreak of COVID-19, the pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an intense search for treatments by the scientific community. In the absence of a vaccine, the goal is to target the viral life cycle and alleviate the lung-damaging symptoms of infection, which can be life-threatening. There are numerous protein kinases associated with these processes that can be inhibited by FDA-approved drugs, the repurposing of which presents an alluring option as they have been thoroughly vetted for safety and are more readily available for treatment of patients and testing in clinical trials. Here, we characterize more than 30 approved kinase inhibitors in terms of their antiviral potential, due to their measured potency against key kinases required for viral entry, metabolism, or reproduction. We also highlight inhibitors with potential to reverse pulmonary insufficiency because of their anti-inflammatory activity, cytokine suppression, or antifibrotic activity. Certain agents are projected to be dual-purpose drugs in terms of antiviral activity and alleviation of disease symptoms, however drug combination is also an option for inhibitors with optimal pharmacokinetic properties that allow safe and efficacious co-administration with other drugs, such as antiviral agents, IL-6 blocking agents, or other kinase inhibitors. [ABSTRACT FROM AUTHOR]

Summary Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. [ABSTRACT FROM AUTHOR]

The article reports on isoxazolidine and related structures as molecules that reconstitute the function of transcriptional activation domains in a cell-free system. The molecules exhibit good activity in a standard in vitro transcription assay comparable to a natural activation domain. Cellular permeability, nuclear localization and cellular stability are not assessed.