The increasing risk of drug resistant bacterial and fungal infections indicate that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis is different in microbes than in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase. Humans do not require this enzyme, and sequence analysis reveals that humans have no homologs for this enzyme. Genetic studies have shown that microorganisms deficient in this enzyme are unable to grow in minimal media, human serum or mice. Furthermore, the gene encoding this enzyme has been identified as a virulence factor and mutations that inactivate the enzyme render microbes non-pathogenic. Despite this biochemical rationale for the development of new antimicrobial agents, little additional biochemical studies have been conducted on N5-CAIR synthetase since its initial discovery, and no drug-like small molecule inhibitors of the enzyme have been reported. To remedy this problem, we will conduct detailed biochemical, mechanistic, structural, computational and drug discovery studies on N5-CAIR synthetase. Our preliminary mechanistic, structural and computational studies have begun to elucidate the mechanism of the enzyme and high-throughput screening has resulted in the identification of drug-like small molecule inhibitors. To continue our studies and accomplish the overall objectives of this application, we will pursue the following three specific aims: 1. Site-directed mutagenesis and pH-rate studies of critical residues proposed to be important in binding and catalysis. 2. Elucidating the mechanism of N5-CAIR synthetase. 3. Understanding, designing, and synthesizing inhibitors of N5-CAIR synthetase. At the end of these studies we expect to have obtained detailed biochemical and mechanistic information on N5-CAIR synthetase and to have developed selective and potent inhibitors of the enzyme.
The incidence of drug-resistant bacterial and fungal infections has increased considerably over the last 20 years and now poses a major challenge for physicians. Invasive fungal and antibiotic-resistant bacterial infections are associated with high mortality rates, increased hospitalizations, and higher health care costs. Unfortunately, methods to treat fungal infections are limited, and treatment options for antibiotic- resistant bacterial infections, while better, are becoming compromised at an alarming rate. This, coupled with the reduction of antimicrobial research within the pharmaceutical industry, highlights the critical need for the continued development of new antimicrobial agents. This grant seeks to address this significant public health problem by studying an enzyme involved in de novo purine biosynthesis. At the conclusion of this study, we anticipate that we will have obtained critical biochemical information about this enzyme and will have identified potent and selective inhibitors of this enzyme. This information will be invaluable for the development of new antimicrobial agents that are capable of treating microbial diseases refractory to existing treatments.
| Dewal, Mahender B; Firestine, Steven M (2013) Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase. Biochemistry 52:6559-67 |
| Dewal, Mahender B; Wani, Amit S; Vidaillac, Celine et al. (2012) Thieno[2,3-d]pyrimidinedione derivatives as antibacterial agents. Eur J Med Chem 51:145-53 |
| Fawaz, Maria V; Topper, Melissa E; Firestine, Steven M (2011) The ATP-grasp enzymes. Bioorg Chem 39:185-91 |
| Thoden, James B; Holden, Hazel M; Paritala, Hanumantharao et al. (2010) Structural and functional studies of Aspergillus clavatus N(5)-carboxyaminoimidazole ribonucleotide synthetase . Biochemistry 49:752-60 |
| Vooturi, S K; Firestine, S M (2010) Synthetic membrane-targeted antibiotics. Curr Med Chem 17:2292-300 |
