Kibdelomycin and kibdelomycin A are antibiotics biosynthesized by Kibdelosporangium banguiense CA-240109. Kibdelomycin demonstrates broad-spectrum activity, exhibiting significant activity against antibiotic-resistant Gram-positive bacteria. These compounds inhibit DNA GyrB and ParE through a distinct U-shaped multi-contact binding mechanism and do not display cross-resistance with established antibiotics. To investigate structure-activity relationships, various methodologies were employed to identify new congeners, including the implementation of repeat batch fermentation. This study presents the discovery, isolation, structural elucidation, and antibacterial assessment of two mono des-chloro congeners of kibdelomycin A produced via repeat batch fermentation. The newly discovered compounds displayed inhibitory effects on bacterial growth in Staphylococcus aureus and Escherichia coli, with minimum inhibitory concentrations (MIC) ranging from 16 to 64 μg/mL. The MIC values for E. coli are comparable to kibdelomycin, whereas for S. aureus the MIC is 64 times less potent than that of kibdelomycin. AI-assisted docking studies involving DNA gyrase B enzymes provide reasonable support for varying activities of the congeners.

Kibdelomycin and amycolamicin are identical antibiotics produced by Kibdelosporangium banguiense CA-240109 and Amycolatopsis sp. MK575-fF5, respectively. They are broad-spectrum antibiotics showing potent activity against antibiotic-resistant Gram-positive bacteria. These compounds inhibit DNA gyrase subunit B (GyrB) and topoisomerase IV (ParE) by a unique U-shaped multicontact binding mode and have no cross-resistance to known antibiotics. Kibdelomycin represents a promising scaffold for developing new antibiotics, but its poor production, structural complexity, and chemical stability limit its chemical modification. In this work, we describe the improvement of kibdelomycin production via strain mutagenesis and the elucidation of kibdelomycin biosynthesis using isotope-labeled feeding experiments and sequencing of the kibdelomycin and amycolamicin biosynthetic gene clusters (BGCs), which are similar but not identical. These studies provide avenues for structure-guided biosynthetic analogue development as well as material for chemical modification.

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.

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.

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.

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.

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).

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.

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.

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 2.3-8.9 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.

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.

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.

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 μ M , two (⩽0.5 μ m ) and three (⩽10 μ m ). These two derivatives showed MIC values ⩽2.5 μ m against most of the 20 MDR strains regardless their resistance profile. Specifically, these lack cross-resistance to rifampicin, isoniazid and moxifloxacin.

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.

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.

ObjectivesPlatensimycin (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.MethodsWe 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.ResultsThe 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.ConclusionsThese 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.

Studies aimed at modulating the contribution of de novo lipogenesis (DNL) to lipid homeostasis are of interest. For example, there appears to be an increased contribution of DNL in cases of dyslipidemia and adipose accretion suggesting that the inhibition of fatty acid synthesis may affect clinical phenotypes. Unfortunately, it is unclear whether inhibition of one specific step in the lipogenic pathway is more important than another. Heavy water (D2O) is generally considered to be a reliable isotopic tracer for quantifying DNL. In contrast to other tracers, D2O yields a homogeneous and quantifiable precursor labeling. However, a drawback of using D2O centers on the relatively long half‐life of water implying that one must wait a substantial time before performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL especially in cases where one aims to use subjects as their own controls. We previously demonstrated the ability to perform back‐to‐back studies of glucose and cholesterol flux by administering multiple doses of D2O and therein circumvent an extensive washout time; presumably these “tricks” can help minimize resources and decrease the turnaround time when testing hypotheses. Herein we now demonstrate the ability to perform back‐to‐back studies of DNL using D2O. However, we have identified special circumstances that limit some aspects of the data analysis, i.e. it is possible to obtain what appear to be negative values for DNL. Using a rodent model we have identified the physiological mechanism that explains the data. We show that one can use D2O to test inhibitors of DNL by performing back‐to‐back studies in higher species (i.e. treat non‐human primates with platensimycin, an inhibitor of fatty acid synthase).Support or Funding InformationAll authors were employed by Merck while this work was being done.

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.

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. (R)-Hydroxy-1,5-naphthyridinone left-hand side (LHS) oxabicyclooctane linked pyridoxazinone right-hand side (RHS) containing NBTIs showed a potent Gram-positive antibacterial profile. SAR around the RHS moiety, including substitutions around pyridooxazinone, pyridodioxane, and phenyl propenoids has been described. A fluoro substituted pyridoxazinone showed an MIC against Staphylococcus aureus of 0.5 μg/mL with reduced functional hERG activity (IC50 333 μM) and good in vivo efficacy [ED90 12 mg/kg, intravenous (iv) and 15 mg/kg, oral (p.o.)]. A pyridodioxane-containing NBTI showed a S. aureus MIC of 0.5 μg/mL, significantly improved hERG IC50 764 μM and strong efficacy of 11 mg/kg (iv) and 5 mg/kg (p.o.). A phenyl propenoid series of compounds showed potent antibacterial activity, but also showed potent hERG binding activity. Many of the compounds in the hydroxy-tricyclic series showed strong activity against Acinetobacter baumannii, but reduced activity against Escherichia coli and Pseudomonas aeruginosa. Bicyclic heterocycles appeared to be the best RHS moiety for the hydroxy-tricyclic oxabicyclooctane linked NBTIs.