Larotrectinib, a selective TRK tyrosine kinase inhibitor (TKI), has demonstrated histology-agnostic efficacy in patients with TRK fusion-positive cancers. While responses to TRK inhibition can be dramatic and durable, duration of response may eventually be limited by acquired resistance. LOXO-195 is a novel, selective TRK TKI designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to TRK TKIs. Activity against these acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first two patients with TRK fusion-positive cancers that developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-Å resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S 2 subsite, and do not bind in the caspase primary aspartic acid binding pocket (S 1 ). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

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Bacterial enoyl-ACP reductase (FabI) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis and is an attractive target for the development of novel antibacterial agents. Previously we reported the development of FabI inhibitor 4 with narrow spectrum antimicrobial activity and in vivo efficacy against Staphylococcus aureus via intraperitoneal (ip) administration. Through iterative medicinal chemistry aided by X-ray crystal structure analysis, a new series of inhibitors has been developed with greatly increased potency against FabI-containing organisms. Several of these new inhibitors have potent antibacterial activity against multidrug resistant strains of S. aureus, and compound 30 demonstrates exceptional oral (po) in vivo efficacy in a S. aureus infection model in rats. While optimizing FabI inhibitory activity, compounds 29 and 30 were identified as having low micromolar FabK inhibitory activity, thereby increasing the antimicrobial spectrum of these compounds to include the FabK-containing pathogens Streptococcus pneumoniae and Enterococcus faecalis. The results described herein support the hypothesis that bacterial enoyl-ACP reductases are valid targets for antibacterial agents.

The MICs of triclosan for 31 clinical isolates of Staphylococcus aureus were 0.016 g/ml (24 strains), 1 to 2 g/ml (6 strains), and 0.25 g/ml (1 strain). All the strains for which triclosan MICs were elevated (>0.016 g/ml) showed three-to fivefold increases in their levels of enoyl-acyl carrier protein (ACP) reductase (FabI) production. Furthermore, strains for which triclosan MICs were 1 to 2 g/ml overexpressed FabI with an F204C alteration. Binding studies with radiolabeled NAD ؉ demonstrated that this change prevents the formation of the stable triclosan-NAD ؉ -FabI complex, and both this alteration and its overexpression contributed to achieving MICs of 1 to 2 g/ml for these strains. Three novel, potent inhibitors of FabI (50% inhibitory concentrations, <64 nM) demonstrated up to 1,000-fold better activity than triclosan against the strains for which triclosan MICs were elevated. None of the compounds tested from this series formed a stable complex with NAD ؉ -FabI. Consequently, although the overexpression of wild-type FabI gave rise to an increase in the MICs, as expected, overexpression of FabI with an F204C alteration did not cause an additional increase in resistance. Therefore, this work identifies the mechanisms of triclosan resistance in S. aureus, and we present three compounds from a novel chemical series of FabI inhibitors which have excellent activities against both triclosan-resistant and -sensitive clinical isolates of S. aureus.Initially, it was thought that the mode of antibacterial action of triclosan was via a general disruptive effect on bacterial membranes (15). However, it has now been shown that the mode of action of triclosan is via inhibition of enoyl-acyl carrier protein (ACP) reductase (FabI) (11) in organisms such as Staphylococcus aureus and Escherichia coli that rely on this enzyme to perform the ultimate step in the elongation cycle of bacterial fatty acid biosynthesis (3,4,5,11,16). Resistance to triclosan has been the subject of much discussion in recent years, and laboratory studies with E. coli and S. aureus have shown that mutations in FabI (G93V/S and G23S, respectively) and their overexpression cause decreases in susceptibility to triclosan (3,4,5,11,16). However, characterization of the triclosan resistance in clinically derived isolates of either organism has not yet been reported.Triclosan exhibits exquisite activity against S. aureus and is used to control the carriage of methicillin-resistant S. aureus in hospitals (1). However, despite debate on the use of triclosan, few surveys have evaluated the level of resistance to triclosan in this important pathogen. Furthermore, the mechanism of triclosan resistance in clinically derived isolates of S. aureus has not been reported. Consequently, in this work we have identified a set of triclosan-resistant isolates of S. aureus and elucidated the mechanisms that give rise to reduced susceptibility to triclosan. Furthermore, we present some novel inhibitors of FabI which are active against these triclosan-resistant isolates of ...

Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.

Bacterial enoyl-ACP reductase (FabI) catalyzes the final step in each cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. Our efforts to identify potent, selective FabI inhibitors began with screening of the GlaxoSmithKline proprietary compound collection, which identified several small-molecule inhibitors of Staphylococcus aureus FabI. Through a combination of iterative medicinal chemistry and X-ray crystal structure based design, one of these leads was developed into the novel aminopyridine derivative 9, a low micromolar inhibitor of FabI from S. aureus (IC(50) = 2.4 microM) and Haemophilus influenzae (IC(50) = 4.2 microM). Compound 9 has good in vitro antibacterial activity against several organisms, including S. aureus (MIC = 0.5 microg/mL), and is effective in vivo in a S. aureus groin abscess infection model in rats. Through FabI overexpressor and macromolecular synthesis studies, the mode of action of 9 has been confirmed to be inhibition of fatty acid biosynthesis via inhibition of FabI. Taken together, these results support FabI as a valid antibacterial target and demonstrate the potential of small-molecule FabI inhibitors for the treatment of bacterial infections.