Repurposing Hsp90 inhibitors as antimicrobials targeting two-component systems identifies compounds leading to loss of bacterial membrane integrity.

The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors. IMPORTANCE: The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae.

Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from E. cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (±0.05) mm, kcat of 6254.72 min-1 and Vmax 0.2 (±0.002) mm·min-1 at 55 °C and acidic pH. Crystal structures of Px_EclTre were determined in the ligand-free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand-free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate-binding site. A cluster of five tryptophans lined the -1 substrate-binding subsite, interacted with crucial active site residues and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.

Functional characterization of Helicoverpa armigera trehalase and investigation of physiological effects caused due to its inhibition by Validamycin A formulation.

Trehalase catalyzes hydrolysis of trehalose and plays a crucial role in insect metabolism. In the present study, phylogenetic analysis and multiple sequence alignment suggested that H. armigera trehalase-1 (HaTre-1) is closely related to other soluble trehalases with conserved signature features and functional sites. We have expressed and purified recombinant HaTre-1 having Vmax ~0.16mM/min and KM ~1.34mM. Inhibition kinetics and Microscale thermophoresis illustrated competitive inhibition of HaTre-1 by Validamycin A having Ki ~3nM and KD ~542nM, respectively. Docking studies of HaTre-1 with Validamycin A indicated that it binds at the active site with multiple hydrogen bonds. Ingestion of Validamycin A resulted in impediment of H. armigera growth and developmental defects. Treated larvae showed concentration dependent decrease in fecundity. It also led to total inhibition of ex-vivo trehalase activity and down-regulation of gene expression of HaTre-1. Relatively high insect mortality was observed on tomato plants sprayed with combination of Validamycin A with Azadirachta indica (neem) and Pongamia pinnata (karanj) oil as compared to the individual treatments. This report has re-emphasized trehalase inhibition as a potential insecticidal strategy and also recommends Validamycin A as a prospective value-added ingredient to commercial bio-pesticide formulations.

Mechanistic insight in potential dual role of sinigrin against Helicoverpa armigera.

The adverse effect of glucosinolates on diverse phytophagous insects is well documented, but its impact on insect physiology has remained enigmatic. Here we report insights into detrimental effects of plant glucosinolate molecule, sinigrin, on Helicoverpa armigera growth and development. In-silico screening of multiple glucosinolates predicted sinigrin as one of the potential inhibitor of H. armigera cathepsin B and L. Insects fed on sinigrin containing diet showed significantly reduced growth (20-30%), delayed pupation (10-15%), decreased fecundity (50-80%) and developmental abnormalities. Further, sinigrin showed 50-60% inhibition of ex-vivo cathepsin like activity which might be a reason for growth and development related abnormalities. In-vitro and mass spectrometry studies highlighted the cytotoxicity caused due to the hydrolysis of sinigrin, into toxic isothiocyanates, in presence of H. armigera whole body extract. In conclusion, insect cathepsin inhibition and isothiocyanate mediated cytotoxicity lead to the dual adverse effect of sinigrin on H. armigera.

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression.

BACKGROUND: Chilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments. METHODS: Recombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength. RESULTS: Larval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested. CONCLUSIONS: Of the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum. GENERAL SIGNIFICANCE: Ingestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.