Antimalarial drugs have thus far been chiefly derived from two sources—natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.
The development of new nonnucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) active against the drug-induced mutations in RT continues to be a very important goal of AIDS research. We used a known inhibitor of HIV-1 RT, 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-alpha]benzimidazole (TZB), as the lead structure for drug design with the objective of making more potent inhibitors against both wild-type (WT) and variant RTs. A series of structurally related 1,2-substituted benzimidazoles was synthesized and evaluated for their ability to inhibit in vitro polymerization by HIV-1 WT RT. A structure-activity study was carried out for the series of compounds to determine the optimum groups for substitution of the benzimidazole ring at the N1 and C2 positions. The best inhibitor, 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimida zole (35), has an IC50 = 200 nM against HIV-1 WT RT in an in vitro enzyme assay. Cytoprotection assays utilizing HIV-infected MT-4 cells revealed that 35 had strong antiviral activity (EC50 = 440 nM) against wild-type virus while retaining broad activity against many clinically observed HIV-1 strains resistant to nonnucleoside inhibitors. Overall, the activity of 35 against wild-type and resistant strains with amino acid substitution in RT is 4-fold or greater than that of TZB and is comparable to that of other nonnucleoside inhibitors currently undergoing clinical trials, most of which do not have the capacity to inhibit the variant forms of the enzyme.
A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.
With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy.
Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum antibacterial activity, including quinolone-resistant isolates, but suffer from potent hERG inhibition (IC(50)= 3 μM for 1). We now disclose the finding that new analogues of 1 with an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition (IC(50)= 31 μM for 24m) and higher free fraction than 1. One optimized analogue, compound 24l, showed moderate clearance in the dog and promising efficacy against Staphylococcus aureus in a mouse thigh infection model.
A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and fastidious Gram-negative bacteria. Here, we describe a series of N-linked oxazolidinone substituents on the benzisoxazole that improve upon the antibacterial activity of initially described compounds of the class, show favorable PK properties, and demonstrate efficacy in an in vivo Staphylococcus aureus infection model. Inhibition of the topoisomerases DNA gyrase and topoisomerase IV from both Gram-positive and a Gram-negative organisms was demonstrated. Compounds showed a clean in vitro toxicity profile, including no genotoxicity and no bone marrow toxicity at the highest evaluated concentrations or other issues that have been problematic for some fluoroquinolones. Compound 1u was identified for advancement into human clinical trials for treatment of uncomplicated gonorrhea based on a variety of beneficial attributes including the potent activity and the favorable safety profile.
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