Identification of and Structural Insights into Hit Compounds Targeting N-Myristoyltransferase for Cryptosporidium Drug Development

Abstract

Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counterscreened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.

Exploring Subsite Selectivity withinPlasmodium vivaxN-Myristoyltransferase Using Pyrazole-Derived Inhibitors

“…1 H NMR (CD 3 OD, 400 MHz, δ, ppm) 8.91 (1H, brs), 8.28 (1H, brs), 8.21−8.17 (1H, m), 8.13 (1H, dd, J = 5.3, 0.6 Hz), 7.43 (1H, dd, J = 8.2, 4.7 Hz), 7.35 (1H, ddd, J = 8.3, 7.4, 1.7 Hz), 7.29 (1H, dd, J = 7.6, 1.7 Hz), 7.14−7.10 (1H, m), 7.02 (1H, dd, J = 7.5, 1.0 Hz), 6.99 (1H, brs), 6.82 (1H, dd, J = 5.2, 1.3 Hz), 4.23 (2H, t, J = 6.7 Hz), 3.83 (3H, s), 3.81−3.76 (4H, m), 3.33−3.29 (4H, m), 3.14 (2H, t, J = 6.6 Hz), 2.04 (3H, s). 13 (…”

“…1 H NMR (CD 3 OD, 400 MHz, δ, ppm) 8.13 (1H, dd, J = 5.3, 0.7 Hz), 7.36 (1H, ddd, J = 8.3, 7.4, 1.8 Hz), 7.31 (1H, dd, J = 7.6, 1.7 Hz), 7.10−7.07 (1H, m), 7.03 (1H, td, J = 7.5, 1.0 Hz), 6.97−6.96 (1H, m), 6.81 (1H, dd, J = 5.2, 1.3 Hz), 4.14 (2H, t, J = 6.6 Hz), 3.80−3.75 (4H, m), 3.74 (3H, s), 3.66 (4H, brs), 3.58 (2H, brs), 3.51 (2H, brs), 3.32−3.30 (4H, m), 2.91 (2H, t, J = 6.6 Hz), 2.03 (3H, s). 13 (1,5-Dimethyl-4- (2-(2-(2-(piperazin-1-yl)pyridin-4-yl)phenoxy)ethyl)-1H-pyrazole-3-carbonyl)glycine (8c). Following General Procedure D, 5c (56 mg, 0.13 mmol) was reacted with 2-(4-tertbutoxycarbonylpiperazin-1-yl)pyridine-4-boronic acid pinacol ester (67 mg, 0.17 mmol), the crude was purified by reversed-phase chromatography (6 g C18 column; ACN in water 0−100%).…”

“…1 H NMR (CD 3 OD, 400 MHz, δ, ppm) 8.09 (1H, dd, J = 5.4, 0.5 Hz), 7.43−7.38 (1H, m), 7.34 (1H, dd, J = 7.7, 1.7 Hz), 7.17−7.14 (1H, m), 7.05 (2H, td, J = 7.5, 1.0 Hz), 6.92 (1H, dd, J = 5.4, 0.5 Hz), 4.20 (2H, t, J = 6.5 Hz), 3.86−3.82 (4H, m), 3.80 (3H, s), 3.78 (2H, s), 3.42−3.38 (4H, m), 3.10 (2H, t, J = 6.5 Hz), 2.04 (3H, s). 13…”

“…vivax contain approximately 5400 genes. , Within this genetic framework, identifying indispensable drug targets such as NMT that directly or indirectly span various phases of the Plasmodium life cycle through their biochemical activity, would constitute significant progress. NMT is now an established drug target for combating malaria and other infectious diseases caused by protozoan parasites, − with crystal structure platforms for structure-aided inhibitor development against Cryptosporidium parvum, Leishmania donovani and major , , and P. vivax, already available.…”

“…8,9 Within this genetic framework, identifying indispensable drug targets such as NMT that directly or indirectly span various phases of the Plasmodium life cycle through their biochemical activity, would constitute significant progress. NMT is now an established drug target for combating malaria and other infectious diseases caused by protozoan parasites, 10−12 structure-aided inhibitor development against Cryptosporidium parvum, 13 Leishmania donovani and major, 14,15 and P. vivax, 16 already available. NMT cotranslationally transfers the myristate moiety of myristoyl-coenzyme A (MyrCoA) to Gly2 of protein substrates after removal of the N-terminal methionine.…”