Project description:A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as β-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that β-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with β-tubulin harboring Cys239, but not β3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in β3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in β-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.
Project description:This study provides comprehensive characterization of the mode of action of bistramide A and identifies structural requirements of bistramide-based compounds that are responsible for severing actin filaments and inhibiting growth of cancer cells in vitro and in vivo. We rationally designed and assembled a series of structural analogs of the natural product, including a fluorescently labeled conjugate. We used TIRF microscopy to directly observe actin filament severing by this series of small molecules, which established that the combination of the spiroketal and the amide subunits was sufficient to enable rapid actin filament disassembly in vitro. In addition, we demonstrated that the enone subunit of bistramide A is responsible for covalent modification of the protein in vitro and in A549 cells, resulting in further increase in the cytotoxicity of the natural product. Our results demonstrate that bistramide A elicits its potent antiproliferative activity by a dual mechanism of action, which entails both severing of actin filaments and covalent sequestration of monomeric actin in the cell.
Project description:We describe the development of a novel anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound is efficacious in a mouse model of visceral leishmaniasis, and has suitable physicochemical, pharmacokinetic and toxicological properties for further development and has been declared a preclinical candidate. Detailed mode of action studies indicate that compounds from this series act principally by inhibiting the parasite protein kinases.
Project description:Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described. Although hydroxamic acids are utilized universally in the development of metalloenzyme inhibitors, they are considered to be poor pharmacophores with reduced activity in vivo. We developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics experiments validate that the cysteine residue is covalently appended with the TAP promoiety. SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy represents an original prodrug design with a dual mode of action for HDAC inhibition.
Project description:Due to the rise of drug resistant forms of tuberculosis there is an urgent need for novel antibiotics to effectively combat these cases and to shorten treatment regimens. Recently, drug screens using whole cell analyses have shown to be successful. However, current high throughput screens focus mostly on stricto sensu life-death screening that give little qualitative information and often require the lengthy process of target and mode of action (MoA) identification. In doing so, promising compound scaffolds or non-optimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early TB drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to sub-inhibitory concentrations of antibiotics with known targets: ciprofloxacin, ethambutol, isoniazid, streptomycin and rifampicin. The resulting dataset comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint i.e. DNA damage for ciprofloxacin and ribosomal stress for streptomycin. Notably, this fingerprint was more distinctive in M. marinum and we decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed for DNA damage, cell wall damage and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so we could identify the putative mode of action for three novel compounds, which confirms our approach.
Project description:Due to the rise of drug resistant forms of tuberculosis there is an urgent need for novel antibiotics to effectively combat these cases and to shorten treatment regimens. Recently, drug screens using whole cell analyses have shown to be successful. However, current high throughput screens focus mostly on stricto sensu life-death screening that give little qualitative information and often require the lengthy process of target and mode of action (MoA) identification. In doing so, promising compound scaffolds or non-optimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early TB drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to sub-inhibitory concentrations of antibiotics with known targets: ciprofloxacin, ethambutol, isoniazid, streptomycin and rifampicin. The resulting dataset comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint i.e. DNA damage for ciprofloxacin and ribosomal stress for streptomycin. Notably, this fingerprint was more distinctive in M. marinum and we decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed for DNA damage, cell wall damage and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so we could identify the putative mode of action for three novel compounds, which confirms our approach.
Project description:Lipoproteins are characterized by a fatty acid moiety at their amino-terminus through which they are anchored into membranes. They fulfill a variety of essential functions in bacterial cells, such as cell wall maintenance, virulence, efflux of toxic elements including antibiotics, and uptake of nutrients. The posttranslational modification process of lipoproteins involves the sequential action of integral membrane enzymes and phospholipids as acyl donors. In recent years, the structures of the lipoprotein modification enzymes have been solved by X-ray crystallography leading to a greater insight into their function and the molecular mechanism of the reactions. The catalytic domains of the enzymes are exposed to the periplasm or external milieu and are readily accessible to small molecules. Since the lipoprotein modification pathway is essential in proteobacteria, it is a potential target for the development of novel antibiotics. In this review, we discuss recent literature on the structural characterization of the enzymes, and the in vitro activity assays compatible with high-throughput screening for inhibitors, with perspectives on the development of new antimicrobial agents.
Project description:To elucidate the antivirulent lactone U1 mode of action, next generation sequencing was applied to analyze the transcriptome of NCTC 8325 cells treated with either compound or DMSO as control.
Project description:To elucidate the antivirulent hydroxy amide (R*,R*)-3 mode of action, next generation sequencing was applied to analyze the transcriptome of NCTC 8325 cells treated with either compound or DMSO as control.
Project description:The mode of action of a representative active compound was investigated using an unbiased metabolomics approach, which has previously been shown to reveal both novel and established modes of action of antimalarials (Creek et al 2016, DOI: 10.1128/AAC.01226-16). The active antimalarial OSM-S-313, and the inactive analogue OSM-S-291, were incubated with trophozoite stage P. falciparum parasites for five hours alongside reference compounds including atovaquone (ATV), chloroquine (CQ), dihydroartemisisin (DHA) and three PfATP4 inhibitors, MMV00073, MMV397264 and MMV390482. Metabolomics analysis of cell pellets and spent media allowed reproducible detection of diverse metabolites from a range of metabolic pathways, with the most significant OSM-S-313-induced perturbations observed within peptide, lipid and energy metabolism, suggesting a specific impact on parasite metabolism.