Optimization of Benzoxazole-Based Inhibitors of Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase

Abstract

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.

Validation of IMP Dehydrogenase Inhibitors in a Mouse Model of Cryptosporidiosis

“…Compounds were evaluated for enzyme inhibition and antiparasitic activity against a reporter Toxoplasma gondii strain (T. gondii/ CpIMPDH) engineered to rely on CpIMPDH for the production of guanine nucleotides (54). Compounds that performed well in these two assays, with 50% inhibitory concentrations (IC 50 ) of Յ20 nM and EC 50 s of Յ2 M, were candidates for testing in the IL-12 knockout mouse model of acute cryptosporidiosis.…”

“…The monolayer was washed with PBS after 3 h and incubated with fresh Ultraculture medium with or without test compounds, inhibitor and media were refreshed after 24 h, and the parasites were cultured for a total of 48 h. Cultures were fixed and counted using an anti-C. parvum fluoresceinlabeled monoclonal antibody (C3C3-fluorescein isothiocyanate [FITC]) or a high-content imaging assay (54). The 50% effective concentration (EC 50 ) values were calculated using the Hill-Slope model using Prism v5 (GraphPad Software Inc., La Jolla, CA): % growth ϭ (maximum Ϫ minimum)/{1 ϩ (EC 50 …”

“…Moreover, the CpIMPDH gene was obtained from an epsilonproteobacterium by lateral gene transfer and thus is very different from host IMPDHs (30,43). We have developed several structurally distinct classes of CpIMPDH inhibitors, achieving nanomolar potency and selectivity in excess of 1,000-fold versus the human enzymes (44)(45)(46)(47)(48)(49)(50). Here, we report the antiparasitic activity of our eight most promising compounds in the interleukin-12 (IL-12) knockout mouse model.…”

Mechanistic Study on the Formation of Compounds from Thioureas

“…Benzoxazoles are a class of heterocyclic compounds exhibiting therapeutical activities (Figure 2), such as, antifungal agents [38][39][40], cytotoxic compounds [41], as anti-inflammatory agents [42], as HIV-1 protease inhibitor [43], as an antibiotic [44], as CpIMPDH inhibitors [45], nonnucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) [46], and antitumour agents [47].…”

Development of novel anti-infective drugs targeting microbial proteins

“…Out of these, MPA and mizoribine have also shown activity against fungal IMPDH, however, they cannot be used for treatment of fungal infections due to their immunosuppressant activity (90,111,112) which would be counterproductive in the case of a fungal infection. Apart from these drugs, several inhibitors targeting human, bacterial and parasitic IMPDH are currently under investigation or in development (113)(114)(115)(116)(117)(118)(119)(120). In conclusion, one of the major challenges is to design and develop inhibitors selective for C. neoformans IMPDH, which do not inhibit both human IMPDH isoforms, type I and type II.…”