Open Source Antibiotics: Simple Diarylimidazoles Are Potent against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) is widely acknowledged as one of the most serious public health threats facing the world, yet the private sector finds it challenging to generate much-needed medicines. As an alternative discovery approach, a small array of diarylimidazoles was screened against the ESKAPE pathogens, and the results were made publicly available through the Open Source Antibiotics (OSA) consortium (https://github.com/opensourceantibiotics). Of the 18 compounds tested (at 32 µg/mL), 15 showed >90% growth inhibition activity against methicillin-resistant Staphylococcus aureus (MRSA) alone. In the subsequent hit-to-lead optimization of this chemotype, 147 new heterocyclic compounds containing the diarylimidazole and other core motifs were synthesized and tested against MRSA, and their structure-activity relationships were identified. While potent, these compounds have moderate to high intrinsic clearance and some associated toxicity. The best overall balance of parameters was found with OSA_975, a compound with good potency, good solubility, and reduced intrinsic clearance in rat hepatocytes. We have progressed toward the knowledge of the molecular target of these phenotypically active compounds, with proteomic techniques suggesting TGFBR1 is potentially involved in the mechanism of action. Further development of these compounds toward antimicrobial medicines is available to anyone under the licensing terms of the project.

SGC-CLK-1: A chemical probe for the Cdc2-like kinases CLK1, CLK2, and CLK4.

Small molecule modulators are important tools to study both basic biology and the complex signaling of protein kinases. The cdc2-like kinases (CLK) are a family of four kinases that have garnered recent interest for their involvement in a diverse set of diseases such as neurodegeneration, autoimmunity, and many cancers. Targeted medicinal chemistry around a CLK inhibitor hit identified through screening of a kinase inhibitor set against a large panel of kinases allowed us to identify a potent and selective inhibitor of CLK1, 2, and 4. Here, we present the synthesis, selectivity, and preliminary biological characterization of this compound - SGC-CLK-1 (CAF-170). We further show CLK2 has the highest binding affinity, and high CLK2 expression correlates with a lower IC50 in a screen of multiple cancer cell lines. Finally, we show that SGC-CLK-1 not only reduces serine arginine-rich (SR) protein phosphorylation but also alters SR protein and CLK2 subcellular localization in a reversible way. Therefore, we anticipate that this compound will be a valuable tool for increasing our understanding of CLKs and their targets, SR proteins, at the level of phosphorylation and subcellular localization.

Dual Inhibition of Mycobacterium tuberculosis and the Host TGFBR1 by an Anilinoquinazoline.

Tuberculosis (TB) control is complicated by the emergence of drug resistance. Promising strategies to prevent drug resistance are the targeting of nonreplicating, drug-tolerant bacterial populations and targeting of the host, but inhibitors and targets for either are still rare. In a cell-based screen of ATP-competitive inhibitors, we identified compounds with in vitro activity against replicating Mycobacterium tuberculosis (Mtb), and an anilinoquinazoline (AQA) that also had potent activity against nonreplicating and persistent Mtb. AQA was originally developed to inhibit human transforming growth factor receptor 1 (TGFBR1), a host kinase that is predicted to have host-adverse effects during Mtb infection. The structure-activity relationship of this dually active compound identified the pyridyl-6-methyl group as being required for potent Mtb inhibition but a liability for P450 metabolism. Pyrrolopyrimidine (43) emerged as the optimal compound that balanced micromolar inhibition of nonreplicating Mtb and TGFBR1 while also demonstrating improved metabolic stability and pharmacokinetic profiles.

Which Small Molecule? Selecting Chemical Probes for Use in Cancer Research and Target Validation.

Small-molecule chemical "probes" complement the use of molecular biology techniques to explore, validate, and generate hypotheses on the function of proteins in diseases such as cancer. Unfortunately, the poor selection and use of small-molecule reagents can lead to incorrect conclusions. Here, we illustrate examples of poor chemical tools and suggest best practices for the selection, validation, and use of high-quality chemical probes in cancer research. We also note the complexity associated with tools for novel drug modalities, exemplified by protein degraders, and provide advice and resources to facilitate the independent identification of appropriate small-molecule probes by researchers. SIGNIFICANCE: Validation of biological targets and pathways will be aided by a shared understanding of the criteria of potency, selectivity, and target engagement associated with small-molecule reagents ("chemical probes") that enable that work. Interdisciplinary collaboration between cancer biologists, medicinal chemists, and chemical biologists and the awareness of available resources will reduce misleading data generation and interpretation, strengthen data robustness, and improve productivity in academic and industrial research.

Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series modulating lysosome function.

The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOMEscan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes. Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.

Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivo S. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.

Kinase Inhibitor Screening Displayed ALK as a Possible Therapeutic Biomarker for Gastric Cancer.

Despite advances in cancer chemotherapy, gastric cancer (GC) continues to have high recurrence rates and poor prognosis with limited treatment options. Understanding the etiology of GC and developing more effective, less harmful therapeutic approaches are vital and urgent. Therefore, this work describes a novel kinase target in malignant gastric cells as a potential therapeutic strategy. Our results demonstrate that among 147 kinase inhibitors (KI), only three molecules were significantly cytotoxic for the AGP-01 cell line. Hence, these three molecules were further characterized in their cellular mode of action. There was significant cell cycle impairment due to the expression modulation of genes such as TP53, CDKN1A, CDC25A, MYC, and CDK2 with subsequent induction of apoptosis. In fact, the Gene Ontology analysis revealed a significant enrichment of pathways related to cell cycle regulation (GO:1902749 and GO:1903047). Moreover, the three selected KIs significantly reduced cell migration and Vimentin mRNA expression after treatment. Surprisingly, the three KIs share the same target, ALK and INSR, but only the ALK gene was found to have a high expression level in the gastric cancer cell line. Additionally, lower survival rates were observed for patients with high ALK expression in TCGA-STAD analysis. In summary, we hypothesize that ALK gene overexpression can be a promising biomarker for prognosis and therapeutic management of gastric adenocarcinoma.

Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas.

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

Chemical Probes for Understudied Kinases: Challenges and Opportunities.

Over 20 years after the approval of the first-in-class protein kinase inhibitor imatinib, the biological function of a significant fraction of the human kinome remains poorly understood while most research continues to be focused on few well-validated targets. Given the strong genetic evidence for involvement of many kinases in health and disease, the understudied fraction of the kinome holds a large and unexplored potential for future therapies. Specific chemical probes are indispensable tools to interrogate biology enabling proper preclinical validation of novel kinase targets. In this Perspective, we highlight recent case studies illustrating the development of high-quality chemical probes for less-studied kinases and their application in target validation. We spotlight emerging techniques and approaches employed in the generation of chemical probes for protein kinases and beyond and discuss the associated challenges and opportunities.

Discovery of a Potent Dual SLK/STK10 Inhibitor Based on a Maleimide Scaffold.

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

NEK5 activity regulates the mesenchymal and migratory phenotype in breast cancer cells.

PURPOSE: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer. METHODS: Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis. RESULTS: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively. CONCLUSION: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression.

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes.

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.

Kinase inhibitor screening reveals aurora-a kinase is a potential therapeutic and prognostic biomarker of gastric cancer.

Gastric cancer is one of the most common and deadly types of cancer in the world, and poor prognosis with treatment failure is widely reported in the literature. In this context, kinases have been considered a relevant choice for targeted therapy in gastric cancer. Here, we explore the antiproliferative and antimigratory effects of the AURKA inhibitor and the prognostic and therapeutic value as a biomarker of gastric cancer. A total of 145 kinase inhibitors were screened to evaluate the cytotoxic or cytostatic effects in the gastric cancer cell line. Using the Alamar Blue assay, flow cytometry, quantitative polymerase chain reaction, and observation of caspase 3/7 activity and cell migration, we investigated the antiproliferative, proapoptotic, and antimigratory effects of the AURKA inhibitor. Moreover, AURKA overexpression was evaluated in the gastric cell lines and the gastric tumor tissue. Out of the 145 inhibitors, two presented the highest antiproliferative effect. Both molecules can induce apoptosis by the caspases 3/7 pathway in addition to inhibiting cancer cell migration, mainly the AURKA inhibitor. Moreover, molecular docking analysis revealed that GW779439X interacts in the active site of the AURKA enzyme with similar energy as a well-described inhibitor. Our study identified AURKA overexpression in the gastric cancer cell line and gastric tumor tissue, revealing that its overexpression in patients with cancer is correlated with low survival. Therefore, it is feasible to suggest AURKA as a potential marker of gastric cancer, besides providing robust information for diagnosis and estimated survival of patients. AURKA can be considered a new molecular target used in the prognosis and therapy of gastric cancer.

Towards a RIOK2 chemical probe: cellular potency improvement of a selective 2-(acylamino)pyridine series.

RIOK2 is an understudied kinase associated with a variety of human cancers including non-small cell lung cancer and glioblastoma. No potent, selective, and cell-active chemical probe currently exists for RIOK2. Such a reagent would expedite re-search into the biological functions of RIOK2 and validate it as a therapeutic target. Herein, we describe the synthesis of naphthyl-pyridine based compounds that have improved cellular activity while maintaining selectivity for RIOK2. While our compounds do not represent RIOK2 chemical probes, they are the best available tool molecules to begin to characterize RIOK2 function in vitro.

A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics.

Over one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines.

Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells.

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.

Targeting Never-In-Mitosis-A Related Kinase 5 in Cancer: A Review.

Mitotic kinases have integral roles in cell processes responsible for cancer development and progression in all tumor types and are common targets for therapeutics. However, a large subset of the human kinome remains unexplored with respect to functionality in cancer systems. Within the mitotic kinases, the never-in-mitosis kinase (NEK) family is emerging as novel kinase targets in various cancer types. NEK5 is an understudied member of the NEK family. While there are more recent studies describing the physiologic function of NEK5, its role in cancer biology remains widely understudied. However, emerging studies implicate that NEK5 has potentially crucial functions in various solid tumors. In this review, we discuss current knowledge regarding the role of NEK5 in cancer and the implications of NEK5 expression and activity in tumor development and metastasis. We summarize current studies that examine NEK5 activity in diverse cancer systems and cellular processes. As an understudied kinase, there are currently no selective NEK5-targeting agents to test the effects of pharmacologic inhibition on cancer, although there exist recent advancements in this area. Here we also include an update on efforts to develop selective pharmacologic inhibition of NEK5, and we discuss the current direction of NEK5-targeting therapeutic development. The generation of selective NEK5 inhibitors is promising new targeted therapies for cancer growth and metastasis.

The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Targeting aurora kinases as a potential prognostic and therapeutical biomarkers in pediatric acute lymphoblastic leukaemia.

Aurora kinases (AURKA and AURKB) are mitotic kinases with an important role in the regulation of several mitotic events, and in hematological malignancies, AURKA and AURKB hyperexpression are found in patients with cytogenetic abnormalities presenting a unfavorable prognosis. The aim of this study was evaluated the mRNA expression profile of pediatric Acute Lymphoblastic Leukaemia (ALL) patients and the efficacy of two AURKA and AURKB designed inhibitors (GW809897X and GW806742X) in a leukemia cell line as a potential novel therapy for ALL patients. Cellular experiments demonstrated that both inhibitors induced cell death with caspase activation and cell cycle arrest, however only the GW806742X inhibitor decreased with more efficacy AURKA and AURKB expression in K-562 leukemia cells. In ALL patients both AURKA and AURKB showed a significant overexpression, when compared to health controls. Moreover, AURKB expression level was significant higher than AURKA in patients, and predicted a poorer prognosis with significantly lower survival rates. No differences were found in AURKA and AURKB expression between gene fusions, immunophenotypic groups, white blood cells count, gender or age. In summary, the results in this study indicates that the AURKA and AURKB overexpression are important findings in pediatric ALL, and designed inhibitor, GW806742X tested in vitro were able to effectively inhibit the gene expression of both aurora kinases and induce apoptosis in K-562 cells, however our data clearly shown that AURKB proves to be a singular finding and potential prognostic biomarker that may be used as a promising therapeutic target to those patients.