Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.
Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.
Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10(3) selectivity for the parasite enzyme over human IMPDH2.
Background: IMP dehydrogenase (IMPDH) is an important drug target because of its role in de novo purine nucleotide biosynthesis.Results: First substrate/cofactor- and substrate/inhibitor-bound complexes of bacterial IMPDHs are determined.Conclusion: A new distinct binding mode of the cofactor adenosine moiety is revealed.Significance: This work offers new insights for the design of more potent and selective inhibitors and the evolution of the active site.
Summary
The protozoan parasite Cryptosporidium parvum is a major cause of gastrointestinal disease; no effective drug therapy exists to treat this infection. Curiously, C. parvum IMPDH (CpIMPDH) is most closely related to prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH gene via horizontal transfer. We previously identified inhibitors of CpIMPDH that do not inhibit human IMPDHs. Here we show that these compounds also inhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, and Streptococcus pyogenes, but not from Escherichia coli. Residues Ala165 and Tyr358 comprise a structural motif that defines susceptible enzymes. Importantly, a second generation CpIMPDH inhibitor has bacteriocidal activity on H. pylori but not Escherichia coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. IMPDH is a target for immunosuppressive, antiviral, and anticancer drugs, but, as of yet, has not been exploited for antimicrobial therapy. We have previously reported potent inhibitors of IMPDH from the protozoan parasite Cryptosporidium parvum (CpIMPDH). Many pathogenic bacteria, including Bacillus anthracis, Staphylococcus aureus, and Listeria monocytogenes, contain IMPDHs that should also be inhibited by these compounds. Herein, we present the structure-activity relationships for the inhibition of B. anthracis IMPDH (BaIMPDH) and antibacterial activity of 140 compounds from five structurally distinct compound series. Many potent inhibitors of BaIMPDH were identified (78% with IC50 ≤ 1 μM). Four compounds had minimum inhibitory concentrations (MIC) of less than 2 μM against B. anthracis Sterne 770. These compounds also displayed antibacterial activity against S. aureus and L. monocytogenes.
Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.
Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1-phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure-activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented.
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