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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.

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.

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Though initially beneficial, TMZ- resistance is universal and second-line interventions are an unmet clinical need. Here we took advantage the 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 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. Finally, 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.TeaserTargeting temozolomide mutations in drug resistant glioma via g-quadruplex and splicing modulators with a putative biomarker.

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8+ T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2−/− mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2−/− macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8+ T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer., Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) is highly expressed in several cancers. Here the authors investigate the role of CaMKK2 expression in the tumour microenvironment and show that CaMKK2 expression in tumour-associated macrophages promotes tumour growth by suppressing T cell anti-tumour activity.

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.

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.

Quinoline- and quinazoline-based kinase inhibitors of the epidermal growth factor receptor (EGFR) have been used to target non-small cell lung cancer (NSCLC) and chordomas with varying amounts of success. We designed and prepared compounds to probe several key structural features including an interaction with Asp855 within the EGFR DGF motif and interactions with the active site water network. EGFR target engagement was then evaluated in a cellular assay, with the inhibitors then profiled in representative cellular models of NSCLC and chordomas. In addition, structure–activity relationship insight into EGFR inhibitor design with potent dimethoxyquin(az)olines identified compounds 1 [N-(3-ethynylphenyl)-6,7-dimethoxyquinolin-4-amine], 4 [N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine], and 7 [4-((3-ethynylphenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile]. We also identified 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (compound 18), which is the most potent inhibitor (IC50=310 nm) of the UCH-2 chordoma cell line to date.

We screened a series of 4-anilinoquinolines and 4-anilinoquinazolines and identified novel inhibitors of Mycobacterium tuberculosis (Mtb). The focused 4-anilinoquinoline/quinazoline scaffold arrays yielded compounds with high potency and the identification of 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (34) with an MIC90 value of 0.63–1.25 µM. We also defined a series of key structural features, including the benzyloxy aniline and the 6,7-dimethoxy quinoline ring, that are important for Mtb inhibition. Importantly the compounds showed very limited toxicity and scope for further improvement by iterative medicinal chemistry.

We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.

Although the human genome provides the blueprint for life, most of the proteins it encodes remain poorly studied. This perspective describes how one group of scientists, in seeking new targets for drug discovery, used open science through unrestricted sharing of small molecules to shed light on dark matter of the genome. Starting initially with a single pharmaceutical company before expanding to multiple companies, a precedent was established for sharing published kinase inhibitors as chemical tools. The integration of open science and kinase chemogenomics has supported the study of many new potential drug targets by the scientific community.

We describe the design of a set of inhibitors to investigate the relationship between cyclin G associated kinase (GAK) and epidermal growth factor receptor (EGFR) in chordoma bone cancers. These compounds were characterized both in vitro and using in cell target engagement assays. The most potent chordoma inhibitors were further characterized in a kinome-wide screen demonstrating narrow spectrum profiles. While we observed a direct correlation between EGFR and antiproliferative effects on chordoma, GAK inhibition appeared to have only a limited effect.

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology.

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.

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 (CAMKK2 IC50 = 3 nM), 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. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly 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 aimed at the identification of CAMKK2 chemical probes and clinical candidates

We report the synthesis of several related 4-anilinoquinazolines as inhibitors of cardiac troponin I–interacting kinase (TNNi3K). These close structural analogs of 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-(dimethylamino)-N-methylbenzenesulfonamide (GSK114) provide new understanding of structure–activity relationships between the 4-anilinoquinazoline scaffold and TNNi3K inhibition. Through a small focused library of inhibitors, we observed that the N-methylbenzenesulfonamide was driving the potency in addition to the more traditional quinazoline hinge-binding motif. We also identified a compound devoid of TNNi3K kinase activity due to the addition of a methyl group in the hinge binding region. This compound could serve as a negative control in the study of TNNi3K biology. Small molecule crystal structures of several quinazolines have been solved, supporting observations made about overall conformation and TNNi3K inhibition.

Journal of medicinal chemistry 63(23), 14626 - 14646 (2020). doi:10.1021/acs.jmedchem.0c01174, STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied ���dark��� kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1., Published by ACS, Washington, DC

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates CAMK1, CAMK4, AMPK, and AKT, leading to numerous physiological responses. The deregulation of CAMKK2 is linked to several diseases, suggesting the utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work, we demonstrate that STO-609, frequently described as a selective inhibitor for CAMKK2, potently inhibits a significant number of other kinases. Through an analysis of literature and public databases, we have identified other potent CAMKK2 inhibitors and verified their activities in differential scanning fluorimetry and enzyme inhibition assays. These inhibitors are potential starting points for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

As antibiotic resistance rises, there is a need for strategies such as antibiotic adjuvants to conserve already-established antibiotics. A family of bacterial kinases known as the penicillin-binding-protein and serine/threonine kinase-associated (PASTA) kinases has attracted attention as targets for antibiotic adjuvants for β-lactams. Here, we report that the pyrazolopyridazine GW779439X sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to various β-lactams through inhibition of the PASTA kinase Stk1. GW779439X potentiates β-lactam activity against multiple MRSA and MSSA isolates, including the sensitization of a ceftaroline-resistant isolate to ceftaroline. In silico modeling was used to guide the synthesis of GW779439X derivatives. The presence and orientation of GW779439X's methylpiperazine moiety was crucial for robust biochemical and microbiologic activity. Taken together, our data provide a proof of concept for developing the pyrazolopyridazines as selective Stk1 inhibitors which act across S. aureus isolates.

Hydrogen peroxide (H2O2) is an abundant molecule associated with biological functions and reacts with natural enzymes, such as catalase. Even though direct H2O2 measurement can be used to diagnose pathological conditions, such as infection and inflammation, H2O2 quantification further enables the detection of disease biomarkers in enzyme-linked assays (e.g., ELISA) in which enzymatic reactions may generate or consume H2O2. Such a quantification is often measured optically with organic dyes in biological media that suffer, however, from poor stability. Currently, the optical H2O2 biosensing without organic-dyes in biological media and at low, submicromolar, concentrations has yet to be achieved. Herein, we rationally design biomimetic artificial enzymes based on antioxidant CeO2 nanoparticles that become luminescent upon their Eu3+ doping. We vary systematically their diameter from 4 to 16 nm and study their catalase-mimetic antioxidant activity, manifested as catalytic H2O2 decomposition in aqueous soluti...

Key Clinical Message Acute life‐threatening intoxications with insecticides are rare. We report a case of accidental near‐fatal thiacloprid intoxication with mass spectrometry‐based analytical confirmation. The initial clinical presentation resembled imminent brain death and/or severe postanoxic encephalopathy. Prolonged supportive treatment resulted in full recovery underlining intoxication as an important differential diagnosis in unclear coma.

FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50 = 0.2 nM) inhibitor of CpFIKK catalytic activity. In addition, we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that CpFIKK may not be involved in parasite growth.

The current feed capacity of electrical interconnects is mainly limited by the poor electromigration (EM) resistance of lead free-solder interconnects. Thus, the community investigates alternative interconnect approaches and materials, e.g. all-copper interconnects using copper pastes. EM is an electron scattering based transport of metal atoms, occurring at high current densities, resulting in voids and thus finally leads to an open interconnect. For all-copper interconnects, it is expected that the maximum current density could be increased, while power losses can be mitigated, due to the higher EM resistance and reduced resistivity of copper compared to solder, respectively. However, all-copper interconnects formed by copper pastes applied without pressure result in a porous copper interconnect morphology with unknown EM performance. Thus, the authors report on results of EM tests on nanoporous copper films. The investigated copper paste is especially designed to be used for flip chip interconnects and contains nano and micron sized copper particles. Paste films were performed by doctor blading, followed by a sintering process at 200°C in formic acid. SEM analysis on FIB cross-sections of sintered films revealed the fusion of the nanoparticles to a nanoporous, sponge like morphology with embedded microparticles. Furthermore, a porosity of approximately 30% could be identified. The high porosity results in a non deterministic electrical network with a measured effective resistivity of ~10 μΩ cm, which is five times higher than the one of bulk copper (1.78 μΩ cm). For detailed investigations of the EM characteristics a setup, as well as a specific test sample layout were designed and implemented. The setup is capable to feed a current of up to 40 A to eight serial samples in a temperature range from room temperature to 250°C in ambient atmosphere. The test samples consist of copper lines, relief printed onto a silicon based carrier substrate, to bridge a 200 μm wide gap between two electrochemically deposited copper electrodes. The voltage drop across the copper line can be measured while driving the current through the line, by four point probing. In addition, a platinum based thermocouple (PT1000) is implemented on the carrier substrate beneath the nanoporous copper. It allows to prove the temperature of the nanoporous copper, to derive exact test conditions and the temperature coefficient of resistance of the nanoporous copper. Initial tests of samples exposed to elevated temperatures above 90°C, but without any current applied, resulted in the resistance increase of the nanoporous copper due to oxidation. Accordingly, a photoresist coverage was applied on the copper lines to mitigate the copper oxidation to levels acceptable for EM testing. A series of EM tests were then performed under constant current conditions to derive the EM kinetics and to explore the damage mechanisms. At current densities of 1 MA/cm^2 and a sample temperature of 90°C, the observed ‘time to failure’ of the nanoporous copper was more than six times higher than the one of tin based lead free solder. Furthermore, the authors will summarize their findings of the EM study performed on the nanoporous copper.

The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S. Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.

[Image: see text] Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

The 4-anilinoquinoline and 4-anilinoquinazoline ring systems have been the focus of significant efforts in prior kinase drug discovery programs, which have led to approved medicines. Broad kinome profiles of these compounds have now been assessed with the advent of advanced screening technologies. These ring systems, while originally designed for specific targets including epidermal growth factor receptor (EGFR), but actually display a number of potent collateral kinase targets, some of which have been associated with negative clinical outcomes. We have designed and synthesized a series of 4-anilinoquin(az)olines in order to better understand the structure-activity relationships of three main collateral kinase targets of quin(az)oline-based kinase inhibitors: cyclin G associated kinase (GAK), STE20-like serine/threonine-protein kinase (SLK) and serine/threonine-protein kinase 10 (STK10). This was achieved through a series of quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites and extensive small-molecule X-ray structural analysis.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

Kinase inhibitors comprise a diverse cohort of chemical scaffolds that are active in multiple biological systems. Currently, thousands of eukaryotic kinase inhibitors are commercially available, have well-characterized targets, and often carry pharmaceutically favorable toxicity profiles. Recently, our group disclosed that derivatives of the natural product meridianin D, a known inhibitor of eukaryotic kinases, modulated behaviors of both Gram-positive and Gram-negative bacteria. Herein, we expand our exploration of kinase inhibitors in Gram-negative bacilli utilizing three commercially available kinase inhibitor libraries and, ultimately, identify two chemical structures that potentiate colistin (polymyxin E) in multiple strains. We report IMD-0354, an inhibitor of IKK-β, as a markedly effective adjuvant in colistin-resistant bacteria and also describe AR-12 (OSU-03012), an inhibitor of pyruvate dehydrogenase kinase-1 (PDK-1), as a potentiator in colistin-sensitive strains. This report comprises the first description of the novel cross-reactivity of these molecules.

SGC-GAK-1 (1), a potent, selective, cell-active chemical probe for cyclin G associated kinase (GAK), was rapidly metabolized in mouse liver microsomes by P450 mediated oxidation. 1 displayed rapid clearance in mice, limiting its utility for in vivo studies. All chemical modifications of 1 that improved metabolic stability led to a loss in GAK activity. However, pretreatment of liver microsomes with the irreversible cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) decreased intrinsic clearance of 1. Coadministration of ABT also greatly improved plasma exposure of 1 in mice, supporting its use as a chemical probe to study the in vivo biology of GAK inhibition.

Epidermal growth factor receptor (EGFR) inhibitors have been used to target non-small cell lung cancer (NSCLC) and chordomas with varying amounts of success. We have probed several key structural features including an interaction with Asp855 within the EGFR DGF motif and interactions with the active site water network. The EGFR target engagement was then evaluated in an in-cell assay. Additionally, inhibitors were profiled in representative cellular models of NSCLC and chordomas. In addition to a structure activity relationship insights for EGFR inhibtior design, we also identified a compound (18) that is the most potent inhibitor (IC50 = 310 nM) on the UCH-2 chordoma cell line to date.

Development of an efficient and scalable synthesis of 6-formylindolo[3,2-b]carbazole (FICZ), a naturally-occurring aryl hydrocarbon receptor (AhR) ligand, allowed its biological and physical properties to be studied. FICZ was shown to be the most potent among a series of 6-substituted indolo[3,2-b]carbazoles for activation of AhR in cells. Photostability studies of FICZ revealed a non-enzymatic mechanism for its conversion to a biologically active quinone. These results further support the hypothesis that FICZ is a light-dependent hormone that links sun exposure to regulation of biological pathways in peripheral tissues.

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.

Polymerization of hemoglobin-S (HbS) in the deoxy conformation shortens the lifespan of sickle red blood cells and promotes intravascular and extravascular hemolysis. When sickle red blood cells are lysed intravascularly, HbS is released into the vascular space where it can consume nitric oxide and be oxidized to higher oxidative forms. During these reactions, ferric (Fe3+) HbS is formed, which readily releases heme. The released heme can activate the innate immune pattern recognition receptor toll-like receptor 4 (TLR4) on endothelium, leading to P-selectin expression, NF-ĸB activation, and microvascular stasis in sickle cell disease (SCD) mice with implanted dorsal skin-fold chambers (DSFCs). Heme-induced TLR4 signalling and stasis are blocked by administering hemopexin with the heme. Plasma hemopexin has the highest binding affinity for free heme, rendering it relatively nonreactive and delivering it safely to the liver for endocytosis and degradation by heme oxygenase-1 (HO-1). Due to chronic hemolysis, hemopexin levels are depleted in SCD patients and mice. We previously reported that intravenous hemopexin induces hepatic HO-1 activity and inhibits ongoing, spontaneous stasis in the venules of SCD mice for up to 48 hours. Inhibition of HO-1 activity with tin protoporphyrin (SnPP) and the administration of carbon monoxide (CO) to SnPP-treated mice demonstrated that HO-1 and its reaction product CO play important roles in the protection against stasis. In the current studies, we asked the question how much of the hemopexin-mediated protection is due to preventing heme activation of TLR4 signalling versus induction of HO-1 and CO production. We wondered if hemopexin complexed with heme would be as effective as hemopexin alone in treating a vaso-occlusive crisis (VOC) induced with hemoglobin heme. First, in a stasis prevention model, we intravenously administered different doses of hemopexin in Townes SCD mice with a DSFC one hour prior to a hemoglobin challenge. Briefly, anesthetized mice were placed on an intravital microscopy stage, and 20-24 flowing subcutaneous venules were selected and mapped. After baseline selection of flowing venules and one hour after administration of different doses of hemopexin (0 - 160 mg/kg body weight), microvascular stasis was triggered by infusing hemoglobin (1 µmol/kg). The same vessels were re-examined for stasis (no flow) and percent stasis was calculated. Our results show that hemopexin effectively reduced stasis in a dose responsive manner when delivered one hour prior to the hemoglobin challenge (Figure 1A). Second, we evaluated hemopexin in a VOC acute treatment model by infusing various doses of hemopexin 30 minutes after the intravenous hemoglobin challenge. We found a dose-dependent effect of hemopexin to reduce and resolve microvascular stasis during an acute VOC one hour after hemopexin infusion (Figure 1B). We obtained similar results after inducing stasis with lipopolysaccharide (LPS) or hypoxia-reoxygenation in place of hemoglobin challenge. Third, we evaluated equimolar hemopexin-heme complexes in a VOC acute treatment model by infusing the same doses of hemopexin-heme 40 minutes after the intravenous hemoglobin challenge (1 µmol/kg) to further elucidate the effects of heme scavenging versus HO-1 induction. Our results showed a striking, dose-dependent reduction in stasis, albeit to a detectably lower degree than hemopexin free of heme (Figure 1C). In summary, hemopexin has a dose-dependent effect to prevent and treat vaso-occlusion induced in a mouse model of SCD by hemoglobin, LPS, and hypoxia-reoxygenation. This effect is partially replicated by hemopexin pre-complexed with heme, suggesting that the beneficial effect is not limited to clearance of circulating heme and prevention of TLR4 signalling. The partial effectiveness of hemopexin-heme complex is consistent with our prior published data implicating CO generation by HO-1 as playing a role in the relief of vaso-occlusion by hemopexin. We conclude that hemopexin shows promise for the treatment of VOC. Additional experiments are needed to clarify the activity of hemopexin in clearance-dependent and -independent mechanisms of resolution of vaso-occlusion. Clinical trials of hemopexin are warranted to evaluate its safety, tolerability and potential efficacy in relieving vaso-occlusive pain crisis in patients with SCD. Disclosures Gentinetta: CSL Behring: Current Employment. Vercellotti:CSL Behring: Research Funding. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment. Belcher:Mitobridge-Astellas: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Hillhurst Biopharmaceuticals: Consultancy.

Screening a series of 4-anilinoquinolines and 4-anilinoquinazolines enabled identification of potent novel inhibitors of dengue virus (DENV). Preparation of focused 4-anilinoquinoline/quinazoline scaffold arrays led to the identification of a series of high potency 6-substituted bromine and iodine derivatives. The most potent compound 6-iodo-4-((3,4,5-trimethoxyphenyl)amino)quinoline-3-carbonitrile (47) inhibited DENV infection with an EC50 = 79 nM. Crucially, these compounds showed very limited toxicity with CC50 values >10 µM in almost all cases. This new promising series provides an anchor point for further development to optimize compound properties.

Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.

Summary New strategies are urgently needed to counter the threat to human health posed by drug-resistant fungi. To explore an as-yet unexploited target space for antifungals, we screened a library of protein kinase inhibitors for the ability to reverse resistance of the most common human fungal pathogen, Candida albicans, to caspofungin, a widely used antifungal. This screen identified multiple 2,3-aryl-pyrazolopyridine scaffold compounds capable of restoring caspofungin sensitivity. Using chemical genomic, biochemical, and structural approaches, we established the target for our most potent compound as Yck2, a casein kinase 1 family member. Combination of this compound with caspofungin eradicated drug-resistant C. albicans infection while sparing co-cultured human cells. In mice, genetic depletion of YCK2 caused an ∼3-log10 decline in fungal burden in a model of systemic caspofungin-resistant C. albicans infection. Structural insights and our tool compound's profile in culture support targeting the Yck2 kinase function as a broadly active antifungal strategy.

We demonstrate an innovative approach utilising both fit-for-purpose kinase mini-panels and kinome-wide panels to progress discovery programs in the optimization of inhibitor potency and selectivity. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like Ser/Thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modeling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel X-ray structure confirmed that the inhibitors bound as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity toward mycobacteria and nocardia. We demonstrated that the inhibitors are uniquely potentiated by β-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB's role in sensing cell wall stress. This is the first demonstration in the phylum actinobacteria that some β-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein down-regulation through drug "off-targets" might be relevant for other inhibitors that serendipitously target pseudokinases. [Abstract copyright: Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.]

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G associated kinase (GAK), together with a structurally-related negative control SGC-GAK-1N (14). SGC-GAK-1 is highly selective in a kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent inhibitor of RIPK2 lacking GAK activity. Together, the chemical probe set of 11, 14, and 18 can be used to interrogate the cellular biology of GAK inhibition.