Antibacterial pyrazoles: tackling resistant bacteria.

Bacterial resistance to antibiotics threatens our progress in healthcare, modern medicine, food production and ultimately life expectancy. Antibiotic resistance is a global concern, which spreads rapidly across borders and continents due to rapid travel of people, animals and goods. Derivatives of metabolically stable pyrazole nucleus are known for their wide range of pharmacological properties, including antibacterial activities. This review highlights recent reports of pyrazole derivatives targeting different bacterial strains focusing on the drug-resistant variants. Pyrazole derivatives target different metabolic pathways of both Gram-positive and Gram-negative bacteria.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells.

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Exploring boron applications in modern agriculture: A structure-activity relationship study of a novel series of multi-substitution benzoxaboroles for identification of potential fungicides.

Several boron-containing small molecules have been approved by the US FDA to treat human diseases. We explored potential applications of boron-containing compounds in modern agriculture by pursuing multiple research and development programs. Here, we report a novel series of multi-substitution benzoxaboroles (1-36), a compound class that we recently reported as targeting geranylgeranyl transferase I (GGTase I) and thereby inhibiting protein prenylation (Kim et al., 2020). These compounds were designed, synthesized, and tested against the agriculturally important fungal pathogens Mycosphaerella fijiensis and Colletotrichum sublineolum in a structure-activity relationship (SAR) study. Compounds 13, 28, 30, 34 and 36 were identified as active leads with excellent antifungal MIC95 values in the range of 1.56-3.13 ppm against M. fijiensis and 0.78-3.13 ppm against C. sublineolum.

Targeting the Mevalonate Pathway in Cancer.

The mevalonate synthesis inhibitors, statins, are mainstay therapeutics for cholesterol management and cardiovascular health. Thirty years of research have uncovered supportive roles for the mevalonate pathway in numerous cellular processes that support oncogenesis, most recently macropinocytosis. Central to the diverse mechanisms of statin sensitivity is an acquired dependence on one mevalonate pathway output, protein geranylgeranylation. New chemical prenylation probes and the discovery of a novel geranylgeranyl transferase hold promise to deepen our understanding of statin mechanisms of action. Further, insights into statin selection and the counterproductive role of dietary geranylgeraniol highlight how we should assess statins in the clinic. Lastly, rational combination strategies preview how statins will enter the oncology toolbox.

Investigation of New Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) by Virtual Screening with Antibacterial Assessment.

BACKGROUND: Considering the interesting role in the peptidoglycan biosynthesis pathway, the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase is an attractive target to develop new antibacterial agents. It catalyzes the first key step of this pathway and its inhibition leads to bacterial cell death. Fosfomycin is known as the natural inhibitor of MurA. OBJECTIVE: The study aimed to introduce new inhibitors of MurA by virtual screening of different chemical compounds libraries, and test the best scored "virtual hits" against three pathogenic bacteria: Escherichia coli, Bacillus subtilis and Staphylococcus aureus. METHODS: A virtual screening of the structural analogues of fosfomycin downloaded from the Pub- Chem database was performed. Moreover, French National Chemical Library and ZINC database were also utilized to identify new structures different from fosfomycin. FlexX was the software used for this study. The antibacterial testing was divided into two methods: disk diffusion and broth dilution. RESULTS: A set of virtual hits was found to have better energy score than that of fosfomycin, seven of them were tested in vitro. In addition, the disk diffusion method explored four compounds that exhibited antibacterial activity: CID-21680357 (fosfomycin analogue), AB-00005001, ZINC04658565, and ZINC901335. The testing was continued by broth dilution method for both compounds CID-21680357 and ZINC901335 to determine their minimum inhibitory concentrations, and ZINC901335 had the best value with 457µg/ml against Staphylococcus aureus. CONCLUSION: Four compounds were found and proven in silico and in vitro to have antibacterial activity, namely CID-21680357, AB-00005001, ZINC04658565, and ZINC901335.

Coenzyme Q biosynthesis inhibition induces HIF-1α stabilization and metabolic switch toward glycolysis.

Coenzyme Q10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Potential Fatty Acid as Antibacterial Agent Against Oral Bacteria of Streptococcus mutans and Streptococcus sanguinis from Basil (Ocimum americanum): In vitro and In silico Studies.

BACKGROUND: Streptococcus mutans and Streptococcus sanguinis are Gram-positive bacteria that cause dental caries. MurA enzyme acts as a catalyst in the formation of peptidoglycan in bacterial cell walls, making it ideal as an antibacterial target. Basil (Ocimum americanum) is an edible plant that is diverse and has been used as a herbal medicine for a long time. It has been reported that basil has a pharmacological effect as well as antibacterial activity. The purpose of this study was to identify antibacterial compounds in O. americanum and analyze their inhibition activity on MurA enzyme. METHODS: Fresh leaves from O. americanum were extracted with n-hexane and purified by a combination of column chromatography on normal and reverse phases together with in vitro bioactivity assay against S. mutans ATCC 25175 and S. sanguinis ATCC 10556, respectively, while in silico molecular docking simulation of lauric acid (1) was conducted using PyRx 0.8. RESULTS: The structure determination of antibacterial compound by spectroscopic methods resulted in an active compound lauric acid (1). The in vitro evaluation of antibacterial activity in compound 1 showed Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of 78.13 and 156.3 ppm and 1250 and 2500 ppm against S. sanguinis and S. mutans, respectively. Further analysis and in silico evaluation determined lauric acid (1) as MurA Enzyme inhibitor. Lauric acid (1) showed a binding affinity of -5.2 Kcal/mol, which was higher than fosfomycin. CONCLUSION: Lauric acid showed the potential as a new natural antibacterial agent through MurA inhibition in bacterial cell wall biosynthesis.

Organoarsenicals inhibit bacterial peptidoglycan biosynthesis by targeting the essential enzyme MurA.

Trivalent organoarsenicals such as methylarsenite (MAs(III)) are considerably more toxic than inorganic arsenate (As(V)) or arsenite (As(III)). In microbial communities MAs(III) exhibits significant antimicrobial activity. Although MAs(III) and other organoarsenicals contribute to the global arsenic biogeocycle, how they exert antibiotic-like properties is largely unknown. To identify possible targets of MAs(III), a genomic library of the gram-negative bacterium, Shewanella putrefaciens 200, was expressed in Escherichia coli with selection for MAs(III) resistance. One clone contained the S. putrefaciens murA gene (SpmurA), which catalyzes the first committed step in peptidoglycan biosynthesis. Overexpression of SpmurA conferred MAs(III) resistance to E. coli. Purified SpMurA was inhibited by MAs(III), phenylarsenite (PhAs(III)) or the phosphonate antibiotic fosfomycin but not by inorganic As(III). Fosfomycin inhibits MurA by binding to a conserved residue that corresponds to Cys117 in SpMurA. A C117D mutant was resistant to fosfomycin but remained sensitive to MAs(III), indicating that the two compounds have different mechanisms of action. New inhibitors of peptidoglycan biosynthesis are highly sought after as antimicrobial drugs, and organoarsenicals represent a new area for the development of novel compounds for combating the threat of antibiotic resistance.

Exploring the Potential Inhibition of Candidate Drug Molecules for Clinical Investigation Based on their Docking or Crystallographic Analyses against M. tuberculosis Enzyme Targets.

Tuberculosis (TB) is a devastating disease responsible for millions of humans' deaths worldwide. It is caused by a mycobacterial organism, the tubercle bacillus or Mycobacterium tuberculosis. Although TB can be treated, cured and can be prevented if patients take prescribed medicines, scientists have never come close to wiping it out due to a sharp rise in the incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) mycobacterium strains. Due to long regimen treatment and emergence of MDR and XDR-TB, it is urgent to re-engineer and reposition old drugs for developing new antimycobacterial entities with novel mechanisms of action to achieve effective TB control even against the resistant forms of TB. To combat the dreadful MDR and XDR-TB, potential targets are being extensively searched for the last couple of years for the design and discovery of active potential antitubercular chemotherapeutics. To explore the disease virulence, potential new tubercular target enzymes such as InhA, MmpL3, ATP synthase, DprE1, QcrB and MenA have been taken into consideration in the present study and the structure-based design of the corresponding target inhibitors which are under clinical investigation has been attempted to identify structural features for the discovery of new chemical entities (NCEs) having specificity towards MDR and XDR Mycobacterium tuberculosis (M. tuberculosis).

Potential Allylpyrocatechol Derivatives as Antibacterial Agent Against Oral Pathogen of S. sanguinis ATCC 10,556 and as Inhibitor of MurA Enzymes: in vitro and in silico Study.

BACKGROUND: Streptococcus sanguinis is Gram-positive bacteria that contribute to caries. Many antibacterial agents are resistant against bacteria so that the discovery of new antibacterial agents is a crucial issue. Mechanism of antibacterial agents by disrupting cell wall bacteria is a promising target to be developed. One of the enzymes contributing to the cell wall is MurA enzyme. MurA is an enzyme catalyzing the first step of peptidoglycan biosynthesis in the cell wall formation. Inhibiting MurA is an effective and efficient way to kill the bacteria. Source of bioactive compounds including the antibacterial agent can be found in natural product such as herbal plant. Piper betle L. was reported to contain active antibacterial compounds. However, there is no more information on the antibacterial activity and molecular mechanism of P. betle's compound against S. sanguinis. PURPOSE: The study aims to identify antibacterial constituents of P. betle L. and evaluate their activities through two different methods including in vitro and in silico analysis. MATERIALS AND METHODS: The antibacterial agent was purified by bioactivity-guided isolation with combination chromatography methods and the chemical structure was determined by spectroscopic methods. The in vitro antibacterial activity was evaluated by disc diffusion and dilution methods while the in silico study of a compound binds on the MurA was determined using PyRx program. RESULTS: The antibacterial compound identified as allylpyrocatechol showed inhibitory activity against S. sanguinis with an inhibition zone of 11.85 mm at 1%, together with MIC and MBC values of 39.1 and 78.1 µg/mL, respectively. Prediction for molecular inhibition mechanism of allylpyrocatechols against the MurA presented two allylpyrocatechol derivatives showing binding activity of -5.4, stronger than fosfomycin as a reference with the binding activity of -4.6. CONCLUSION: Two allylpyrocatechol derivatives were predicted to have a good potency as a novel natural antibacterial agent against S. sanguinis through blocking MurA activity that causes disruption of bacterial cell wall.

Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas.

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

Inhibiting Protein Prenylation with Benzoxaboroles to Target Fungal Plant Pathogens.

Fungal pathogens pose an increasing threat to global food security through devastating effects on staple crops and contamination of food supplies with carcinogenic toxins. Widespread deployment of agricultural fungicides has increased crop yields but is driving increasingly frequent resistance to available agents and creating environmental reservoirs of drug-resistant fungi that can also infect susceptible human populations. To uncover non-cross-resistant modes of antifungal action, we leveraged the unique chemical properties of boron chemistry to synthesize novel 6-thiocarbamate benzoxaboroles with broad spectrum activity against diverse fungal plant pathogens. Through whole genome sequencing of Saccharomyces cerevisiae isolates selected for stable resistance to these compounds, we identified mutations in the protein prenylation-related genes, CDC43 and ERG20. Allele-swapping experiments confirmed that point mutations in CDC43, which encodes an essential catalytic subunit within geranylgeranyl transferase I (GGTase I) complex, were sufficient to confer resistance to the benzoxaboroles. Mutations in ERG20, which encodes an upstream farnesyl pyrophosphate synthase in the geranylgeranylation pathway, also conferred resistance. Consistent with impairment of protein prenylation, the compounds disrupted membrane localization of the classical geranylgeranylation substrate Cdc42. Guided by molecular docking predictions, which favored Cdc43 as the most likely direct target, we overexpressed and purified functional GGTase I complex to demonstrate direct binding of benzoxaboroles to it and concentration-dependent inhibition of its transferase activity. Further development of the boron-containing scaffold described here offers a promising path to the development of GGTase I inhibitors as a mechanistically distinct broad spectrum fungicide class with reduced potential for cross-resistance to antifungals in current use.

The mevalonate pathway is a crucial regulator of tendon cell specification.

Tendons and ligaments are crucial components of the musculoskeletal system, yet the pathways specifying these fates remain poorly defined. Through a screen of known bioactive chemicals in zebrafish, we identified a new pathway regulating tendon cell induction. We established that statin, through inhibition of the mevalonate pathway, causes an expansion of the tendon progenitor population. Co-expression and live imaging studies indicate that the expansion does not involve an increase in cell proliferation, but rather results from re-specification of cells from the neural crest-derived sox9a+/sox10+ skeletal lineage. The effect on tendon cell expansion is specific to the geranylgeranylation branch of the mevalonate pathway and is mediated by inhibition of Rac activity. This work establishes a novel role for the mevalonate pathway and Rac activity in regulating specification of the tendon lineage.

Nature-driven approaches to non-natural terpene analogues.

Covering: up to 2019The reactions catalysed by terpene synthases belong to the most complex and fascinating cascade-type transformations in Nature. Although many accept only one natural terpene precursor and convert it with high selectivity into only one product, several of these remarkable biocatalysts were recently shown to have a surprising plasticity towards non-natural substrate analogues. For an easy access to the topic also for readers who are new to the field, this review will first briefly cover the principles of natural terpene biosynthesis. This is followed by a chapter that highlights purely chemical transformations mimicking terpene synthase catalysed reactions. Then, the main focus of this article will shed light on the recent advances of terpene synthase catalysed transformations of synthetic substrate analogues. As will be demonstrated, a simple conceptual approach extensively broadens the chemical space that can be reached with terpene synthases.

The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection.

Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin. Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent CDI.

Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity.

Echinocandin resistance in Candida is a great concern, as the echinocandin drugs are recommended as first-line therapy for patients with invasive candidiasis. However, therapeutic efforts to thwart echinocandin resistance have been hampered by a lack of fungal specific drug targets. Here, we show that deleting CDC43, the ß subunit of geranylgeranyltransferase type I (GGTase I), confers hypersensitivity to echinocandins, which renders GGTase I a tractable target in combatting echinocandin resistance. The membrane localization of Rho1, which is critical for (1,3)-ß-d-glucan synthase Fks1 activation, is disrupted in the cdc43 mutant, resulting in decreased amounts of glucans in the cell wall, thereby exacerbating the cell wall stress upon caspofungin addition. Guided by this insight, we found that selective chemical inhibition of GGTase I by L-269289 potentiates echinocandin activity and renders echinocandin-resistant Candida albicans responsive to treatment in vitro and in animal models for disseminated infection. Furthermore, L-269289 and echinocandins also act in a synergistic manner for the treatment of Candida tropicalis and Candida parapsilosis Importantly, deletion of CDC43 is lethal in Candida glabrata L-269289 is active on its own to kill C. glabrata, and its fungicidal activity is enhanced when combined with caspofungin. Thus, targeting GGTase I has therapeutic potential to address the clinical challenge of echinocandin-resistant candidiasis.

Biosynthesis of heme O in intraerythrocytic stages of Plasmodium falciparum and potential inhibitors of this pathway.

A number of antimalarial drugs interfere with the electron transport chain and heme-related reactions; however, the biosynthesis of heme derivatives in Plasmodium parasites has not been fully elucidated. Here, we characterized the steps that lead to the farnesylation of heme. After the identification of a gene encoding heme O synthase, we identified heme O synthesis in blood stage parasites through the incorporation of radioactive precursors. The presence of heme O synthesis in intraerythrocytic stages of Plasmodium falciparum was confirmed by mass spectrometry. Inabenfide and uniconazole-P appeared to interfere in heme synthesis, accordingly, parasite growth was also affected by the addition of these drugs. We conclude that heme O synthesis occurs in blood stage-P. falciparum and this pathway could be a potential target for antimalarial drugs.

Catalytic Mechanism and Covalent Inhibition of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): Implications to the Design of Novel Antibacterials.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first step in the biosynthesis of the bacterial cell wall. This pathway is essential for the growth of bacteria but missing in mammals, that nominates MurA as an attractive antibacterial target. MurA has a flexible loop whose conformational change is known to be part of the activation mechanism of the enzyme. We have shown that the loop closed conformation makes the proton transfer from Cys115 to His394 possible by a low barrier exothermic process. QM/MM MD simulations revealed that the activated thiolate is able to react with phosphoenolpyruvate (PEP), the natural substrate of MurA. The binding free energy profile of several covalent inhibitors with various warheads reacting with the activated Cys115 was calculated by QM/MM MD simulations and confirmed that reaction barrier heights tend to separate active from inactive compounds. Our results give new insight into the catalytic mechanism and covalent inhibition of MurA and suggest that QM/MM MD simulations are able to support ligand design by providing sensible relative free energy barriers for covalent inhibitors with various warheads reacting with thiolate nucleophiles.

A Phase I Study of GGTI-2418 (Geranylgeranyl Transferase I Inhibitor) in Patients with Advanced Solid Tumors.

BACKGROUND: Geranylgeranyltransferase I (GGTase I) catalyzes geranylgeranylation, a modification required for the function of many oncogenic RAS-related proteins. GGTI-2418 is a peptidomimetic small molecule inhibitor of GGTase I. OBJECTIVE: The aim of this study was to establish the maximum tolerated dose of GGTI-2418 in patients with advanced solid tumors. PATIENTS AND METHODS: This was a phase I, open-label, dose-escalation study conducted in two US centers (University of Pennsylvania and Indiana University) in adults with treatment-refractory advanced solid tumors. An accelerated dose-escalation schema was used across eight dose levels, from 120 to 2060 mg/m2, administered on days 1-5 of each 21-day cycle. RESULTS: Fourteen patients were enrolled in the dose-escalation cohort. No dose-limiting toxicities were observed, and 2060 mg/m2 was determined to be the maximum tolerated dose. The only potential drug-related grade 3 or 4 toxicities were elevated bilirubin and alkaline phosphatase in a single patient with concurrent malignant biliary obstruction. No objective responses were observed. Four of thirteen evaluable patients had stable disease for up to 6.7 months. The study was terminated prior to dose expansion based on a sponsor decision. Pharmacokinetic analysis demonstrated a mean terminal half-life of 1.1 h. CONCLUSIONS: GGTI2418 was safe and tolerable at all tested dose levels with some evidence of disease stability. Due to rapid elimination, dosing of GGTI2418 in this study may have been inadequate to achieve optimal inhibition of its target, GGTase I.