Antibacterial and mechanism of action studies of boxazomycin A.

Boxazomycins A-C are potent broad-spectrum antibiotics isolated from Actinomycetes strain G495-1 in 1987. We now report that boxazomycin A inhibits bacterial growth by selectively inhibiting protein synthesis, its effect is bacteriostatic, and it is equally active against drug resistant bacterial strains. No cross-resistance to protein synthesis inhibitors was observed suggesting that its inhibition is distinct from clinical protein synthesis inhibitors. We also report in vivo efficacy in a Staphylococcus aureus murine infection model supported by corresponding pharmacokinetic studies.

Comparative Evaluation of Multiplex PCR, RLEP PCR and LAMP PCR in Urine, Stool and Blood Samples for the Diagnosis of Pediatric Leprosy - A Cross-Sectional Study.

OBJECTIVE: To compare the diagnostic efficacy of multiplex polymerase chain reaction (PCR), Mycobacterium leprae-specific repetitive element (RLEP) PCR and loop-mediated isothermal amplification (LAMP) PCR in the diagnosis of pediatric leprosy as an alternative to slit-skin smear (SSS) examination. METHODS: A cross-sectional study was performed on 26 children aged 0-18 years with characteristic skin lesions of leprosy. SSS examination for acid fast bacilli (AFB) was performed for all children. Additionally, urine, stool and blood samples were tested by three PCR techniques - multiplex, RLEP and LAMP. The results of these tests were compared with each other and with results of SSS examination for acid fast bacilli (AFB) using appropriate statistical tests. RESULTS: Out of 26 patients studied, SSS examination was positive for AFB in 7 cases (26.9%). In blood samples, the positivity of multiplex PCR, RLEP PCR and LAMP PCR was 84.6%, 80.8%, and 80.8%, respectively. Multiplex PCR in blood samples was positive in 100% (n = 7) of SSS positive cases and 84.2% (16 out of 19) of the SSS negative cases (P < 0.001). The positivity of all PCR methods in urine and stool samples was significantly lesser than in blood. CONCLUSION: Multiplex PCR in blood sample is a superior diagnostic tool for pediatric leprosy compared to RLEP PCR and LAMP PCR as well as SSS examination.

History and Prospects of Drug Discovery and Development Collaboration between Industry and Academia.

Research collaborations and licensing deals are critical for the discovery and development of life-saving drugs. This practice has been ongoing since the inception of the pharmaceutical industry. The current process of drug discovery and development is complex, regulated, and highly regimented, having evolved over time. Academia excels in the discovery of fundamental scientific concepts and biological processes, while industry excels in translational science and product development. Potential for collaboration exists at every step of the drug discovery and development continuum. This perspective walks through such collaborative activities, provides examples, and offers tips for potential collaborations.

Discovery, synthesis, activities, structure-activity relationships, and clinical development of combretastatins and analogs as anticancer drugs. A comprehensive review.

Covering: 1982 to up to the end of 2022Bioassay guided purification of the extracts of Combretum caffrum led to the discovery of six series of combretastatins A-D with cytotoxic activities ranging from sub nM to >50 µM ED50's against a wide variety of cancer cell lines. Of these, cis-stilbenes combretastatins A-4 and A-1 were the most potent, exhibiting in vivo efficacy against a wide variety of tumor types in murine models. These antimitotic agents inhibited tubulin polymerization by reversibly binding to the colchicine binding sites. They inhibited tumor growth by a novel antivascular and antineogenesis mechanism in which they stopped blood flows to the blood vessels causing necrosis. Over 20 clinical trials of the phosphate prodrugs of combretastatin A-4 (CA4P) and A-1 (CA1P) showed objective and stable responses against many tumor types, with increased survival times of many patients along with the confirmed cure of certain patients inflicted with anaplastic thyroid cancers. Medicinal chemistry efforts led to the identification of three new leads (AVE8062, BNC105P, SCB01A) with improved in vitro and in vivo potency and an often-improved cellular spectrum. Unfortunately, these preclinical improvements did not translate clinically in any meaningful way. Objectively, CA4P remained the best compound and has garnered many Orphan drug designations by FDA. Clinical trials with tumor genetic mapping, particularly from previous responders, may help boost the success of these compounds in future studies. A comprehensive review of combretastatin series A-D, including bioassay guided discovery, total syntheses, and structure-activity relationship (SAR) studies, biological and mechanistic studies, and preclinical and clinical evaluations of the isolated combretastatins and analogs, along with the personal perspective of the author who originated this project, is presented.

Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

Nocathiacin, Thiazomycin, and Polar Analogs Are Highly Effective Agents against Toxigenic Clostridioides difficile.

Clostridioides difficile is a commensal Gram-positive gut bacterium that causes C. difficile-associated diarrhea. Currently available antibacterial therapeutic treatment options are effective except for the repeated recurrences significantly burdening the health care system and causing mortality. The development of new therapeutic modalities including new effective antibiotics with a low rate of recurrence has been unpredictive and exceedingly challenging, requiring continued profiling of many new classes of antibiotics. Nocathiacins and thiazomycins are a class of thiazolyl peptides exhibiting potent and selective broad-spectrum Gram-positive activity including activity against the anaerobe C. difficile. These compounds showed MIC values of 0.015-0.06 µg/mL against C. difficile with more than 100-200-fold selectivity versus commensurate Gram-negative Bacteroides fragilis. Nocathiacin I and one of its analogs exhibited potent in vivo efficacy in the gold-standard hamster model of C. difficile infection, providing 100% protection in this lethal model at 6.25 mg/kg orally twice daily. The efficacy was corroborated by robust reduction of cecum C. difficile burden and proportionate exposure of the compounds in the cecum contents without any systemic absorption. In this paper, details of the results of in vitro, in vivo, pharmacodynamics, and pharmacokinetic studies have been described.

Discovery and Development of Dolastatin 10-Derived Antibody Drug Conjugate Anticancer Drugs.

Dolastatin 10 is an extremely potent broad-spectrum antitubulin anticancer pentapeptide isolated from Dolabella auricularia. The two-dimensional structure was elucidated by NMR and mass spectrometric analyses. The absolute configuration was determined by a convergent total synthesis. SAR studies established that modifications at C- and N-terminals were tolerated for cytotoxic activity. Human clinical trials of dolastatin 10 and auristatin PE (a C-terminal analog) showed occasional signs of efficacy but failed due to lack of separation of toxicity and efficacy. Nanomolar cytotoxicity helped transition this class of pentapeptides to the next phase of development as antibody drug conjugates (ADCs) by reducing systemic toxicity. Four ADC drugs (Adcetris, Padcev, Polivy, and Blenrep) carrying monomethyl auristatin E (MMAE, vedotin) and monomethyl auristatin F (MMAF, mafodotin) payloads have been approved for treatment of a number of cancers expressing antibody-specific antigens. More than 36 ADCs carrying a variety of pentapeptide analogues are undergoing preclinical and clinical developments. They are being evaluated in more than 200 human trials. A comprehensive review of the discovery, total synthesis of dolastatin 10 and new amino acids, SAR studies of dolastatin 10 and auristatins, conjugations to antibodies, and preclinical and clinical development of ADCs have been presented.

Pregnane-Oximino-Alkyl-Amino-Ether Compound as a Novel Class of TGR5 Receptor Agonist Exhibiting Antidiabetic and Anti-Dyslipidemic Activities.

INTRODUCTION: The present study deals with the synthesis of pregnane-oximino-amino-alkyl-ethers and their evaluation for antidiabetic and anti-dyslipidemic activities in validated animal and cell culture models. METHODS: The effect on glucose tolerance was measured in sucrose-loaded rats; antidiabetic activity was evaluated in streptozotocin (STZ)-induced diabetic rats and genetically diabetic db/db mice; the anti-dyslipidemic effect was characterized in high-fructose, high-fat diet (HFD)-fed dyslipidemic hamsters. The effect on glucose production and glucose utilization was analyzed in HepG2 liver and L6 skeletal muscle cells, respectively. RESULTS: From the synthesized molecules, pregnane-oximino-amino-alkyl-ether (compound 14b) improved glucose clearance in sucrose-loaded rats and exerted antihyperglycemic activity on STZ-induced diabetic rats. Further evaluation in genetically diabetic db/db mice showed temporal decrease in blood glucose, and improvement in glucose tolerance and lipid parameters, associated with mild improvement in the serum insulin level. Moreover, compound 14b treatment displayed an anti-dyslipidemic effect characterized by significant improvement in altered lipid parameters of the high-fructose, HFD-fed dyslipidemic hamster model. In vitro analysis in the cellular system suggested that compound 14b decreased glucose production in liver cells and stimulated glucose utilization in skeletal muscle cells. These beneficial effects of compound 14b were associated with the activation of the G-protein-coupled bile acid receptor TGR5. CONCLUSION: Compound 14b exhibits antidiabetic and anti-dyslipidemic activities through activating the TGR5 receptor system and can be developed as a lead for the management of type II diabetes and related metabolic complications.

Rapid, Selective, and Sensitive Method for Semitargeted Discovery of Congeneric Natural Products by Liquid Chromatography Tandem Mass Spectrometry.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Identification of cyclic hexapeptides natural products with inhibitory potency against Mycobacterium tuberculosis.

OBJECTIVE: Our aim was to identify natural products with anti-tubercular activity. RESULTS: A set of ~ 500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3 to 8.9 µM. Eleven structural analogs had no significant activity (MIC > 20 µM) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.

Pharmacokinetics of Second-Line Antituberculosis Drugs in Children with Multidrug-Resistant Tuberculosis in India.

We studied the pharmacokinetics of levofloxacin (LFX), pyrazinamide (PZA), ethionamide (ETH), and cycloserine (CS) in children with multidrug-resistant tuberculosis (MDR-TB) who were being treated according to the Revised National TB Control Programme (RNTCP) guidelines in India. This observational, pharmacokinetic study was conducted in 25 children with MDR-TB at the Sarojini Naidu Medical College, Agra, India, who were being treated with a 24-month daily regimen. Serial blood samples were collected after directly observed administration of drugs. Estimations of plasma LFX, PZA, ETH, and CS were undertaken according to validated methods by high-performance liquid chromatography. Adverse events were noted at 6 months of treatment. The peak concentration (Cmax) of LFX was significantly higher in female than male children (11.5 µg/ml versus 7.3 µg/ml; P = 0.017). Children below 12 years of age had significantly higher ETH exposure (area under the concentration-time curve from 0 to 8 h [AUC0-8]) than those above 12 years of age (17.5 µg/ml · h versus 9.4 µg/ml; P = 0.030). Multiple linear regression analysis showed significant influence of gender on Cmax of ETH and age on Cmax and AUC0-8 of CS. This is the first and only study from India reporting on the pharmacokinetics of LFX, ETH, PZA, and CS in children with MDR-TB treated in the Government of India program. More studies on the safety and pharmacokinetics of second-line anti-TB drugs in children with MDR-TB from different settings are required.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

What is an "ideal" antibiotic? Discovery challenges and path forward.

An ideal antibiotic is an antibacterial agent that kills or inhibits the growth of all harmful bacteria in a host, regardless of site of infection without affecting beneficial gut microbes (gut flora) or causing undue toxicity to the host. Sadly, no such antibiotics exist. What exist are many effective Gram-positive antibacterial agents as well as broad-spectrum agents that provide treatment of certain Gram-negative bacteria but not holistic treatment of all bacteria. However effectiveness of all antibacterial agents is being rapidly eroded due to resistance. This viewpoint provides an overview of today's antibiotics, challenges and potential path forward of discovery and development of new (ideal) antibiotics.

Thiazomycin, nocathiacin and analogs show strong activity against clinical strains of drug-resistant Mycobacterium tuberculosis.

Thiazolyl peptides are a class of natural products with potent Gram-positive antibacterial activities. Lack of aqueous solubility precluded this class of compounds from advancing to clinical evaluations. Nocathiacins and thiazomycins are sub-classes of thiazolyl peptides that are endowed with structural features amenable for chemical modifications. Semi-synthetic modifications of nocathiacin led to a series of analogs with improved water solubility, while retaining potency and antibacterial spectrum. We studied the activities of a selection of two natural products (nocathiacin and thiazomycin) as well as seven polar semi-synthetic analogs against twenty clinical strains of Mycobacterium tuberculosis with MDR phenotypes. Two compounds show useful activity against H37Rv strain with MIC values ⩽1 µM, two (⩽0.5 µm) and three (⩽10 µm). These two derivatives showed MIC values ⩽2.5 µm against most of the 20 MDR strains regardless their resistance profile. Specifically, these lack cross-resistance to rifampicin, isoniazid and moxifloxacin.

Correction: The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

[This corrects the article DOI: 10.1371/journal.pone.0164133.].

The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

OBJECTIVES: Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. METHODS: We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. RESULTS: The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. CONCLUSIONS: These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

Microarray analyses for identifying genes conferring resistance to pepper leaf curl virus in chilli pepper (Capsicum spp.).

Pepper leaf curl virus (PepLCV) is a serious threat to pepper (Capsicum spp.) production worldwide. Molecular mechanism underlying pepper plants response to PepLCV infection is key to develop PepLCV resistant varieties. In this study, we generated transcriptome profiles of PepLCV resistant genotype (BS-35) and susceptible genotype (IVPBC-535) after artificial viral inoculation using microarray technology and detail experimental procedures and analyses are described. A total of 319 genes differentially expressed between resistant and susceptible genotypes were identified, out of that 234 unique genes were found to be up-regulated > 2-fold in resistant line BS-35 when compared to susceptible, IVPBC-535. The data set we generated has been analyzed to identify genes that are involved in the regulation of resistance against PepLCV. The raw data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE41131.

In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.

Discovery and development of kibdelomycin, a new class of broad-spectrum antibiotics targeting the clinically proven bacterial type II topoisomerase.

Kibdelomycin is a complex novel antibiotic, discovered by applying a highly sophisticated chemical-genetic Staphylococcus aureus Fitness Test (SaFT) approach, that inhibits the clinically established bacterial targets, gyrase and topoisomerase IV. It exhibits broad-spectrum antibacterial activity against aerobic bacteria including MRSA and Acinetobacter baumannii. It is slowly bactericidal and has a low frequency of resistance. In an anaerobic environment, it exhibits narrow-spectrum activity and inhibits the growth of gut bacteria Clostridium difficile (MIC 0.125µg/mL) without affecting the growth of commensal Gram-negative organisms particularly, Bacteroides sp. It is highly efficacious in the hamster model of C. difficile infection providing 100% protection at >6mg/kg and 80% protection at 1.56mg/kg by oral dosing without systemic exposure. X-ray co-crystal structures of kibdelomycin bound to GyrB and ParE showed a unique dual arm 'U shaped' multisite binding never encountered with any other gyrase inhibitors. Kibdelomycin is poised for preclinical development for C. difficile treatment, and most importantly, the co-crystal structures of kibdelomycin provide unique insight for structure-guided structure modification, which could lead to better broader-spectrum systemic antibiotic potentially covering many ESKAPE pathogens.

Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.