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Summary Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7 as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7 as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition. [ABSTRACT FROM AUTHOR]

Summary The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of low skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in “redhaired” Mc1r- deficient mice, demonstrated significant protection against UV damage. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt-inducible kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CRTC and CREB activity. Here, we describe the development of small-molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r -deficient mice and normal human skin. These findings demonstrate a realistic pathway toward UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk. [ABSTRACT FROM AUTHOR]

Summary Embryonic stem cells (ESCs) can self-renew or differentiate into any cell type, a phenomenon known as pluripotency. Distinct pluripotent states, termed naive and primed pluripotency, have been described. However, the mechanisms that control naive-primed pluripotent transition are poorly understood. Here, we perform a targeted screen for kinase inhibitors, which modulate the naive-primed pluripotent transition. We find that XMD compounds, which selectively inhibit Erk5 kinase and BET bromodomain family proteins, drive ESCs toward primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, we reveal distinct functions for Erk5 and Brd4 in pluripotency regulation. We show that Erk5 signaling maintains ESCs in the naive state and suppresses progression toward primed pluripotency and neuroectoderm differentiation. Additionally, we identify a specialized role for Erk5 in defining ESC lineage selection, whereby Erk5 inhibits a cardiomyocyte-specific differentiation program. Our data therefore reveal multiple critical functions for Erk5 in controlling ESC identity. [ABSTRACT FROM AUTHOR]

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that regulates Antioxidant Response Element (ARE)-mediated transcription of a plethora of antioxidant and protective genes to counteract the harmful effects of reactive oxygen species or environmental carcinogens. Studies have demonstrated that pre-emptive activation of the Nrf2–ARE pathway reinforces the cellular defense mechanism against oxidative stress and leads to protection in a variety of disease models. Non-carcinogenic ARE inducers have been identified from a variety of chemical classes that enhance the transcriptional activity of Nrf2 through S-alkylation of reactive cysteines within the cellular redox sensor protein Keap1 (Kelch-like ECH associated protein 1). Here we review the currently known small molecule ARE inducers and their reported biological activities in various models. [Copyright &y& Elsevier]

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase and the founding member of a signaling pathway that regulates many fundamental features of cell growth and division. In cells, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Together, these complexes coordinate a variety of processes that include protein translation, autophagy, proliferation, survival and metabolism in response to nutrient, energy and growth factor signals. Consistent with its role as a growth-promoting pathway, numerous studies have found that mTOR signaling is hyper-activated in a broad spectrum of human cancers. In particular, mTORC2 is considered a primary effector of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, which is mutated in a majority of human cancers, in part through its ability to phosphorylate and regulate the proto-oncogene Akt/PKB. Many biological functions of mTOR have been pharmacologically explored using the natural product rapamycin, an allosteric inhibitor that has been reviewed extensively elsewhere. This review will focus specifically on the development of small molecule ATP-competitive inhibitors of mTOR and their prospects as a targeted therapy. [Copyright &y& Elsevier]

Covalent inhibitors of the KRASG12C oncoprotein have recently been developed and are being evaluated in clinical trials. Resistance to targeted therapies is common and may limit long-term efficacy of KRAS inhibitors (KRASi). To identify pathways of adaptation to KRASi and predict drug combinations that circumvent resistance, we use mass-spectrometry-based quantitative temporal proteomics to profile the proteomic response to KRASi in pancreatic and lung cancer 2D and 3D cellular models. We quantify 10,805 proteins, representing the most comprehensive KRASi proteome (https://manciaslab.shinyapps.io/KRASi/). Our data reveal common mechanisms of acute and long-term response between KRASG12C-driven tumors. Based on these proteomic data, we identify potent combinations of KRASi with phosphatidylinositol 3-kinase (PI3K), HSP90, CDK4/6, and SHP2 inhibitors, in some instances converting a cytostatic response to KRASi monotherapy to a cytotoxic response to combination treatment. Overall, using quantitative temporal proteomics, we comprehensively characterize adaptations to KRASi and identify combinatorial regimens with potential therapeutic utility. • Quantitative proteomics defines adaptation to oncogenic KRAS inhibitors (KRASi) • Adaptation to KRASi involves cell-cycle reactivation and metabolic rewiring • Combination with PI3Ki, HSP90i, or EGFRi overcomes adaptation to KRASi monotherapy • KRASi with CDK4/6i suppresses growth in 3D culture and warrants further evaluation Santana-Codina et al. use mass-spectrometry-based quantitative temporal proteomics to identify mechanisms of resistance to recently developed oncogenic KRASG12C inhibitors currently undergoing clinical trial evaluation. Using this "KRASi proteome" resource (https://manciaslab.shinyapps.io/KRASi/), they identify combinatorial regimens with PI3K, HSP90, CDK4/6, and SHP2 inhibitors to circumvent resistance, with potential future therapeutic utility. [ABSTRACT FROM AUTHOR]

S1P receptors (S1PR1-5) are a group of GPCRs activated by a high affinity binding with S1P that have important roles in the regulation of the immune system. A potent S1PR agonist FTY720 is an immunomodulator used to treat multiple sclerosis and several 'second generation' drugs are under clinical development. Subtype-selective agonists have been reported for each S1PR isotype, some of which are used as pharmacological tools for functional studies. Here we report the discovery and initial characterization of compound 5c, a benzo[b]thiophene amino carboxylate which exhibits potent and selective agonist activity for S1PR4. Compound 5c has an EC50 = 200 nM as an agonist in GTPγ35S binding assay for S1PR4 and exhibits no activity against S1PR1,2,3,5. We confirmed its potent activity and decent S1PR subtype selectivity using biochemical and cellular assays. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation. A potent and selective CDK7 degrader was developed. JWZ-5-13 induces a rapid and sustained CDK7 degradation and exhibits high proteome-wide selectivity. JWZ-5-13 displays advantages in inhibition of cancer cell proliferation over its parental binder and displays bioavailability in a pharmacokinetic study. [Display omitted] • JWZ-5-13 significantly degrades CDK7 in low nanomolar in multiple cell line. • JWZ-5-13 exhibits high proteome-wide selectivity. • JWZ-5-13 exhibited advantages in inhibition of cancer cell proliferation over its parental binder. • JWZ-5-13 displayed bioavailability in a pharmacokinetic study. [ABSTRACT FROM AUTHOR]

Abstract: A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using a cellular Lck-dependent proliferation assay and are capable of inhibiting Lck at single-digit nanomolar concentrations. This scaffold is likely to serve a valuable template for developing potent inhibitors of a number of SFKs. [Copyright &y& Elsevier]

Abstract: A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to prepare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited single-digit nanomolar IC50s against Lck in biochemical and cellular assays. These 4-amino-6-benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors. [Copyright &y& Elsevier]

Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells. We show that QL47 inhibits protein neosynthesis initiated by both canonical cap-driven and noncanonical initiation strategies, most likely by targeting an early step in translation elongation. Our findings thus establish QL47 as a new small-molecule inhibitor that can be utilized to probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent. [ABSTRACT FROM AUTHOR]

The TAIRE family of kinases are an understudied branch of the CDK kinase family, that have been implicated in a number of cancers. This manuscript describes the design, synthesis and SAR of covalent CDK14 inhibitors, culminating in identification of FMF-04-159-2, a potent, covalent CDK14 inhibitor with a TAIRE kinase biased selectivity profile. [ABSTRACT FROM AUTHOR]

Quinolines and thiazolopyridines were developed as allosteric inhibitors of MALT1, with good cellular potency and exquisite selectivity. Mouse pharmacokinetic (PK) profiling showed these to have low in vivo clearance, and moderate oral exposure. The thiazolopyridines were less lipophilic than the quinolines, and one thiazolopyridine example was active in our hIL10 mouse pharmacodynamic (PD) model upon oral dosing. [ABSTRACT FROM AUTHOR]

Potent and selective substrate-based covalent inhibitors of MALT1 protease were developed from the tetrapeptide tool compound Z-VRPR-fmk. To improve cell permeability, we replaced one arginine residue. We further optimized a series of tripeptides and identified compounds that were potent in both a GloSensor reporter assay measuring cellular MALT1 protease activity, and an OCI-Ly3 cell proliferation assay. Example compounds showed good overall selectivity towards cysteine proteases, and one compound was selected for further profiling in ABL-DLBCL cells and xenograft efficacy models. [ABSTRACT FROM AUTHOR]

Most cancer cells are dependent on a network of deregulated signaling pathways for survival and are insensitive, or rapidly evolve resistance, to selective inhibitors aimed at a single target. For these reasons, drugs that target more than one protein (polypharmacology) can be clinically advantageous. The discovery of useful polypharmacology remains serendipitous and is challenging to characterize and validate. In this study, we developed a non-genetic strategy for the identification of pathways that drive cancer cell proliferation and represent exploitable signaling vulnerabilities. Our approach is based on using a multitargeted kinase inhibitor, SM1-71, as a tool compound to identify combinations of targets whose simultaneous inhibition elicits a potent cytotoxic effect. As a proof of concept, we applied this approach to a KRAS-dependent non-small cell lung cancer (NSCLC) cell line, H23-KRASG12C. Using a combination of phenotypic screens, signaling analyses, and kinase inhibitors, we found that dual inhibition of MEK1/2 and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) is critical for blocking proliferation in cells. Our work supports the value of multitargeted tool compounds with well-validated polypharmacology and target space as tools to discover kinase dependences in cancer. We propose that the strategy described here is complementary to existing genetics-based approaches, generalizable to other systems, and enabling for future mechanistic and translational studies of polypharmacology in the context of signaling vulnerabilities in cancers. [ABSTRACT FROM AUTHOR]

Abstract The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses. Highlights • We have developed a biochemical assay for inhibitors of Zika E protein. • High-throughput screening using this assay led to discovery of seven lead compounds that inhibit Zika and dengue virus entry. • The Zika E inhibitors block the fusion step of viral entry. [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]

[Display omitted] The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. Furthermore, LRRK2 function as a scaffolding protein in several pathways has been implicated as a plausible mechanism underlying neurodegeneration caused by LRRK2 mutations. Given that both the kinase activity and scaffolding function of LRRK2 have been linked to neurodegeneration, we developed proteolysis-targeting chimeras (PROTACs) targeting LRRK2. The degrader molecule JH-XII-03-02 (6) displayed high potency and remarkable selectivity for LRKK2 when assessed in a of 468 panel kinases and serves the dual purpose of eliminating both the kinase activity as well as the scaffolding function of LRRK2. [ABSTRACT FROM AUTHOR]

Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases have proved popular cancer targets and many inhibitors have been developed. The majority of these clinical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants of ligand binding and selectivity. [ABSTRACT FROM AUTHOR]

Through a structure-based drug design approach, a tricyclic benzonaphthyridinone pharmacophore was used as a starting point for carrying out detailed medicinal structure-activity relationhip (SAR) studies geared toward characterization of a panel of proposed BTK inhibitors, including 6 (QL-X-138), 7 (BMX-IN-1) and 8 (QL47). These studies led to the discovery of the novel potent irreversible BTK inhibitor, compound 18 (CHMFL-BTK-11). Kinetic analysis of compound 18 revealed an irreversible binding efficacy ( k inact /K i ) of 0.01 μM −1 s −1 . Compound 18 potently inhibited BTK kinase Y223 auto-phosphorylation (EC 50 < 100 nM), arrested cell cycle in G0/G1 phase, and induced apoptosis in Ramos, MOLM13 and Pfeiffer cells. We believe these features would make 18 a good pharmacological tool for studying BTK-related pathologies. [ABSTRACT FROM AUTHOR]

The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR. [ABSTRACT FROM AUTHOR]

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity, including fibroblasts and invasive cancer cells. However, pathways of MT1-MMP up-regulation are not clearly understood. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it up-regulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation and its physiological relevance are not known. In this study, we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not the β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited the collagen-induced MT1-MMP-dependent activation of pro-MMP-2 and up-regulation of MT1-MMP at the gene and protein levels. Interestingly, DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited the MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell-surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signaling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2activation was found to be noticeably more effective when cells were stimulated by collagen without the non-helical telopeptide region compared with intact collagen fibrils. Furthermore, DDR2-dependent MT1-MMP activation by cartilage was found to be more efficient when the tissue was partially damaged. These data suggest that DDR2 is a microenvironment sensor that regulates fibroblast migration in a collagen-rich environment. [ABSTRACT FROM AUTHOR]

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47's antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC 90 . QL-XII-47's inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity. [ABSTRACT FROM AUTHOR]

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5 , 27 , was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases. [ABSTRACT FROM AUTHOR]

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production. [ABSTRACT FROM AUTHOR]

TAK1 (transforming growth factor-β-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the ‘DFG-motif’ of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, β and γ), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kβ, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ. JWZ-1-80 induces rapid and sustained proteasomal degradation of endogenous PI5P4Kγ through recruitment of the E3 ligase adaptor VHL. JWZ-1-80 exhibits excellent proteome-wide selectivity and does not degrade PI5P4Kα and PI5P4Kβ. [Display omitted] • A first-in-class PI5P4Kγ degrader were developed. • JWZ-1-80 selectively degraded PI5P4Kγwithout affecting PI5P4Kα and PI5P4Kβ. • JWZ-1-80 degraded PI5P4Kγ through the ubiquitin-protease pathway. • JWZ-1-80 exhibits high proteome-wide selectivity. • JWZ-1-80 induced the degradation of PI5P4Kγ in multiple cell lines. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDK) are attractive targets for drug discovery due to their wide range of cellular functions. CDK11 is an understudied CDK with roles in transcription and splicing, cell cycle regulation, neuronal function, and apoptosis. In this study, we describe a medicinal chemistry campaign to identify a CDK11 inhibitor. Employing a promising but nonselective CDK11-targeting scaffold (JWD-047), extensive structure-guided medicinal chemistry modifications led to the identification of ZNL-05-044. A combination of biochemical evaluations and NanoBRET cellular assays for target engagement guided the SAR towards a 2,4-diaminothiazoles CDK11 probe with significantly improved kinome-wide selectivity over JWD-047. CDK11 inhibition with ZNL-05-044 leads to G2/M cell cycle arrest, consistent with prior work evaluating OTS964, and impacts CDK11-dependent mRNA splicing in cells. Together, ZNL-05-044 serves as a tool compound for further optimization and interrogation of the consequences of CDK11 inhibition. [Display omitted] • ZNL-05-044 is a selective CDK11 inhibitor. • Structure-guided design identifies ZNL-05-044 from a multi-targeted scaffold. • Biochemical and NanoBRET assays guide development of ZNL-05-044. • ZNL-05-044 treatment leads to G2/M cell cycle arrest and impacts mRNA splicing. [ABSTRACT FROM AUTHOR]

Her3 is a member of the human epidermal growth factor receptor (EGFR) tyrosine kinase family, and it is often either overexpressed or deregulated in many types of human cancer. Her3 has not been the subject of small-molecule inhibitor development because it is a pseudokinase and does not possess appreciable kinase activity. We recently reported on the development of the first selective irreversible Her3 ligand ( TX1-85-1 ) that forms a covalent bond with cysteine 721 which is unique to Her3 among all kinases. We also developed a bi-functional compound ( TX2-121-1 ) containing a hydrophobic adamantane moiety and the same warhead of TX1-85-1 that is capable of inhibiting Her3-dependent signaling and growth. Here we report on the structure-based medicinal chemistry effort that resulted in the discovery of these two compounds. [ABSTRACT FROM AUTHOR]

[Display omitted] The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg. [ABSTRACT FROM AUTHOR]

The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure–activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 μM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 μM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S 10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent. [ABSTRACT FROM AUTHOR]

Abstract: Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson’s disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC50s of around 10nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3–1.0μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg. [Copyright &y& Elsevier]

Abstract: HG-7-85-01(22) and HG-7-86-01(26) are thiazolo[5,4-b]pyridine containing type II tyrosine kinase inhibitors with potent cellular activity against both wild-type and ‘gatekeeper’ mutant T315I- Bcr-Abl. Here we report on the ‘hybrid design’ concept and subsequent structure activity guided optimization efforts that resulted in the development of these inhibitors. [Copyright &y& Elsevier]

Abstract: Here we describe the synthesis and characterization of a number of 3-amino-1H-indazol-6-yl-benzamides that were designed to target the ‘DFG-out’ conformation of the kinase activation loop. Several compounds such as 4 and 11 exhibit single-digit nanomolar EC50s against FLT3, c-Kit and the gatekeeper T674M mutant of PDGFRα. [Copyright &y& Elsevier]

Abstract: Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson’s disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC50 of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1–0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg. [Copyright &y& Elsevier]

Abstract: Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life. [Copyright &y& Elsevier]

Abstract: Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer. A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M ‘gatekeeper’ resistance mutation relative to wild-type EGFR were discovered by library screening. A representative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-dependent cellular proliferation assays. [ABSTRACT FROM AUTHOR]

Summary: BMK1 is activated by mitogens and oncogenic signals and, thus, is strongly implicated in tumorigenesis. We found that BMK1 interacted with promyelocytic leukemia protein (PML), and inhibited its tumor-suppressor function through phosphorylation. Furthermore, activated BMK1 notably inhibited PML-dependent activation of p21. To further investigate the BMK-mediated inhibition of the tumor suppressor activity of PML in tumor cells, we developed a small-molecule inhibitor of the kinase activity of BMK1, XMD8-92. Inhibition of BMK1 by XMD8-92 blocked tumor cell proliferation in vitro and significantly inhibited tumor growth in vivo by 95%, demonstrating the efficacy and tolerability of BMK1-targeted cancer treatment in animals. [Copyright &y& Elsevier]

Abstract: A series of alkyne-containing type II inhibitors with potent inhibitory activity of T315I Bcr-Abl has been identified. The most active compound 4 exhibits an EC50 of less than 1nM against wild-type Bcr-Abl and an EC50 of 10nM against T315I mutant but is broadly active against a number of other kinases. [Copyright &y& Elsevier]

Abstract: A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties. [Copyright &y& Elsevier]

Abstract: Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter. [Copyright &y& Elsevier]

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of AbI tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH2-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH2-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors. [ABSTRACT FROM AUTHOR]

Abstract: The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC50s of 40nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the ‘DFG-out’ inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors. [Copyright &y& Elsevier]

Abstract: Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and thus as a putative drug for the treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFα production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFα production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFα, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFα and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-γ. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFα production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFα and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases. [Copyright &y& Elsevier]

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torini, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC 1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1. [ABSTRACT FROM AUTHOR]

Abstract: Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications. [Copyright &y& Elsevier]

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. [Display omitted] [ABSTRACT FROM AUTHOR]