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In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China, ultimately leading to a global pandemic (1). Since January 2020, the United States has observed a dramatic [...]

The engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100-Gly), allowing glyphosate to bind in its extended, inhibitory conformation. conformational change | crystal structure | genetic modification | mutation

The crystal structure and kinetic characterization of enzyme MurA from Enterobacter cloacae and Escherichia coli is described. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflected the importance of noncovalent binding potential, even for covalent inhibitors for ensuring inactivation efficiency and specificity.

Combination X-ray crystallography and solution NMR spectroscopy were used to characterize the ferredoxin nicotinamide adenine dinucleotide phosphate reductase from Pseudomonas aeruginosa (pa-FPR) in complex with [NADP.sup.+]. The observations from NMR spectra of pa-FPR and the pa-FPR-NADP complex provided insights into the C'-terminal sequence residues, which does not undergo conformational exchange in the presence or absence of [NADP.sup.+] and the stepwise-electron-proton transfer of hydride in the oxidation of NADPH by FPR enzymes.

The differential inhibition of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzymes by analogues of tetrahedral reaction intermediate (TI) is examined. Results reveal that class II EPSPS are generally less susceptible to inhibition by these TI analogues.

The products of the two genes encoding electron transfer proteins upregulated by Pseudomonas aeruginosa in response to iron starvation are identified as the redox partners of the heme oxygenase expressed under low-iron stress conditions.

Calpain is ubiquitous Ca(super 2+) activated proteolytic enzyme, where a crystal structure of the human mu-calpain protease core (hmuI-II) containing a Gly213Ala mutation and covalently inactivated by a peptidomimetic (ZLLYCH2F). The hydrogen bonding and hydrophobic interaction between active site residues and the inhibitor where the terminal ZL portion of the inhibitor is solvent based in recognition of calpain's natural protein.

The high-resolution structures of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate are determined. The results suggest that the structural events during open-closed transition of EPSPS are changed as a result of inhibitor action.

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDKs) areserine/threonine protein kinasesthat act as key regulatory elements in cell cycle progression. Wedescribe the development of highly potent diaminothiazole inhibitorsof CDK2 (IC50= 0.0009–0.0015 μM) from a singlehit compound with weak inhibitory activity (IC50= 15 μM),discovered by high-throughput screening. Structure-based design wasperformed using 35 cocrystal structures of CDK2 liganded with distinctanalogues of the parent compound. The profiling of compound 51against a panel of 339 kinases revealed high selectivityfor CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4,and CDK6. Compound 51inhibited the proliferation of13 out of 15 cancer cell lines with IC50values between0.27 and 6.9 μM, which correlated with the complete suppressionof retinoblastoma phosphorylation and the onset of apoptosis. Combined,the results demonstrate the potential of this new inhibitors seriesfor further development into CDK-specific chemical probes or therapeutics. [ABSTRACT FROM AUTHOR]

MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity. [ABSTRACT FROM AUTHOR]

Glyphosate ( N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on mechanisms of resistance to glyphosate as obtained through natural diversity, the gene-shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer a rationale for the means by which the modifications made have had their intended effect. [ABSTRACT FROM AUTHOR]

Focuses on the interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in atomic detail. Use of the enzyme in the synthesis of aromatic amino acids and compounds in algae, higher plants, bacteria and fungi; Schematic representation of ligand binding in the EPSP synthase-S3P-glyphosate complex.

Investigates the induced-fit mechanism in Enterobacter cloacae MurA by kinetic studies and X-ray crystallography. Existence of a hydrogen-bonding network mainly supplied by water molecules; Presence of intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding.

Inhibition of cancer-promoting kinases is an established therapeutic strategy for the treatment of many cancers, although resistance to kinase inhibitors is common. One way to overcome resistance is to target orthogonal cancer-promoting pathways. Bromo and Extra-Terminal (BET) domain proteins, which belong to the family of epigenetic readers, have recently emerged as promising therapeutic targets in multiple cancers. The development of multitarget drugs that inhibit kinase and BET proteins therefore may be a promising strategy to overcome tumor resistance and prolong therapeutic efficacy in the clinic. We developed a general computational screening approach to identify novel dual kinase/bromodomain inhibitors from millions of commercially available small molecules. Our method integrated machine learning using big datasets of kinase inhibitors and structure-based drug design. Here we describe the computational methodology, including validation and characterization of our models and their application and integration into a scalable virtual screening pipeline. We screened over 6 million commercially available compounds and selected 24 for testing in BRD4 and EGFR biochemical assays. We identified several novel BRD4 inhibitors, among them a first in class dual EGFR-BRD4 inhibitor. Our studies suggest that this computational screening approach may be broadly applicable for identifying dual kinase/BET inhibitors with potential for treating various cancers. [ABSTRACT FROM AUTHOR]

Resistance to androgen receptor (AR) antagonists is a significant problem in the treatment of castration-resistant prostate cancers (CRPC). Identification of the mechanisms by which CRPCs evade androgen deprivation therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family, epigenetically promotes HOXB13 expression. Consistently, genetic disruption of HOXB13 or pharmacological suppression of its mRNA and protein expression by the novel dual-activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation and cell migration, and suppression of CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell-cycle progression, nucleotide metabolism, and chromatin assembly. Notably, although the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT-treated CRPC cell lines and patient-derived circulating tumor cells (CTC) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlates with HOXB13 expression in CTCs of mCRPC patients who did not respond to abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13-positive metastatic prostate cancers. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core proproliferative network driving ADT resistance that is targetable with potent dual-activity bromodomain-kinase inhibitors., (©2018 American Association for Cancer Research.)

Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775's anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.

Meiotic recombination ensures faithful segregation of homologous chromosomes during meiosis and generates genetic diversity in gametes. MEIOB (meiosis specific with OB domains), a meiosis-specific single-stranded DNA-binding homolog of replication protein A1 (RPA1), is essential for meiotic recombination. Here, we investigated the molecular mechanisms of MEIOB by characterizing its binding partners spermatogenesis associated 22 (SPATA22) and RPA. We find that MEIOB and SPATA22 form an obligate complex and contain defined interaction domains. The interaction between these two proteins is unusual in that nearly any deletion in the binding domains abolishes the interaction. Strikingly, a single residue D383 in MEIOB is indispensable for the interaction. The MEIOB/SPATA22 complex interacts with the RPA heterotrimeric complex in a collaborative manner. Furthermore, MEIOB and SPATA22 are recruited to induced DNA double-strand breaks (DSBs) together but not alone. These results demonstrate the cooperative property of the MEIOB-SPATA22 complex in its interaction with RPA and recruitment to DSBs., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.)

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) constitute the small enzyme family of enolpyruvyl transferases, which catalyze the chemically unusual reaction of enolpyruvyl transfer. MurA catalyzes the first step in the biosynthesis of the bacterial cell wall; AroA is the sixth enzyme of the shikimate pathway leading to the synthesis of aromatic compounds in numerous microorganisms and plants. Because both metabolic pathways are absent from mammals but essential for the growth of microorganisms, MurA and AroA are attractive targets for the development of novel antimicrobial drugs. We have determined the x-ray structures of the D305A mutant of Enterobacter cloacae MurA and the D313A mutant of Escherichia coli AroA, both of which crystallized in the presence of their substrates. The structures depict the tetrahedral reaction intermediate states of the enzymes and prove that, without the aspartate side chain, the overall addition-elimination reaction in both enzymes is halted after the addition step. The presented structures lead to a new view of the catalytic mechanism and, moreover, provide an ideal starting point for the rational design of potent inhibitors of MurA and AroA.