Spiropyrimidinetrione DNA Gyrase Inhibitors with Potent and Selective Antituberculosis Activity.

New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new spiropyrimidinetriones (SPTs), DNA gyrase inhibitors having activity against drug-resistant (), the causative agent of TB. While the clinical candidate zoliflodacin has progressed to phase 3 trials for the treatment of gonorrhea, compounds herein demonstrated higher inhibitory potency against DNA gyrase (e.

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in .

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 μM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds.

Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies.

Phenotypic screening of a Medicines for Malaria Venture compound library against () identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of suggested a novel mechanism of action. The compounds were bactericidal against replicating and retained potency against clinical isolates of .

Synthesis, Structure-Activity Relationship, and Mechanistic Studies of Aminoquinazolinones Displaying Antimycobacterial Activity.

Phenotypic whole-cell screening against () in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone which was optimized for solubility by replacing the sulfone moiety with a sulfoxide . The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound displayed favorable properties and was therefore selected for pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone .

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs.

Binding of MgtR, a Salmonella transmembrane regulatory peptide, to MgtC, a Mycobacterium tuberculosis virulence factor: a structural study.

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system.

Experimental malaria infection triggers early expansion of natural killer cells.

In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic-stage Plasmodium chabaudi. Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 h postinfection, followed by strong increases in natural killer (NK) cell-associated and major histocompatibility complex class I-related transcripts by 32 h postinfection. We showed by flow cytometry that the observed elevation in NK cell-associated transcripts was the result of a dramatic increase in the proportion of NK cells in the blood during infection.

Plasmodium falciparum: genome wide perturbations in transcript profiles among mixed stage cultures after chloroquine treatment.

A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P.

Regulatory motifs uncovered among gene expression clusters in Plasmodium falciparum.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum.

Validation of microsatellite markers for use in genotyping polyclonal Plasmodium falciparum infections.

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples.

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum.

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites.

Short report: Dynamics of Plasmodium falciparum malaria after sub-optimal therapy in Uganda.

We followed parasite genotypes of 75 patients for 42 days after treatment of uncomplicated malaria with chloroquine + sulfadoxine-pyrimethamine in Kampala, Uganda. Infections were complex (mean, 2.88 strains) and followed three patterns: 27% of patients eliminated all strains and remained parasite-free, 48% had a long aparasitemic interval followed by reappearance of original strains after 3-33 days (mean, 9.

In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal.

To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus.

Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal.

Mapping of the Plasmodium falciparum multidrug resistance gene 5'-upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites.

The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasite's response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug-resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed.