Structural determinants of inhibitor selectivity in prokaryotic IMP dehydrogenases

Abstract

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 E. coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.

Figure 1. The IMPDH reaction

a. Chemical mechanism: a conserved Cys attacks C2 of IMP and hydride is transferred to NAD⁺ producing the covalent intermediate E-XMP*. E-XMP* is hydrolyzed with a conserved Arg residue acting as a general base to produce XMP. b. The hydride transfer reaction proceeds in an open enzyme conformation. After NADH departs, a mobile flap folds into the NAD site, carrying the catalytic Arg into the active site. Inhibitors compete with the flap, so the equilibrium between open and closed states is a determinant of inhibitor affinity. c. Phylogenetic tree of IMPDHs. Constructed as described in (Min, et al., 2008).

Figure 2. Structures of IMPDH inhibitor binding sites

a. Structure of C64. b. Structure of the CpIMPDH•IMP•C64 complex (salmon; PDB accession 3KHJ (MacPherson, et al.)) and resistant IMPDHs from T. foetus (green; 1LRT (Gan, et al., 2002)) and Chinese hamster (blue; 1JR1, nearly identical to human IMPDH2 (Sintchak, et al., 1996)). Residues within 5 Å of C64 are displayed. C64 is shown in gray with a transparent surface; CpIMPDH residues are labeled; residue from the adjacent monomer are denoted with a '. Residues Thr221 and Glu329 are hidden under C64. c. The surface of the NAD binding site rendered by conservation of residues in prokaryotic IMPDHs from T. foetus, C. parvum, H. pylori, B. burgodorferi, S. pyogenes and EcIMPDH. Dark magenta, 100% conserved; tan, 63%; dark cyan, 25%. The NAD analog tiazofurin adenine dinucleotide (TAD) is light gray. The tiazofurin binding site is circled in blue and the ADP binding site is circled in red. d. The surface of the C64 binding site rendered by conservation of residues in sensitive IMPDHs from C. parvum, H. pylori, and B. burgdorferi, as well as the S250A/L444Y variant of EcIMPDH. Ser164, Met326 and Ser354 are within 5 Å of C64; Val143 is 8 Å away. Dark magenta, 100% conserved; tan, 60%; dark cyan, 20%. C64 depicted in ball-and-stick in light gray and IMP is shown in stick in dark gray. e. The surface of the C64 binding site rendered by conservation of residues in CpIMPDH and IMPDHs from 19 pathogenic bacteria: Acinetobacter baumannii (wound infection), Bacillus anthracis (anthrax), Bacteroides fragilis (peritoneal infections), Brucella abortus/melitensis/suis (brucellosis), B. burgdorferi (Lyme disease), Burkholderia cenocepacia (infection in cystic fibrosis), Bu. mallei (glanders), Bu. pseudomallei (melioidosis), Campylobacter jejuni (food poisoning), C. lari (food poisoning), Coxiella burnetii (Q fever), Francisella tularensis (tularemia), H. pylori (gastric ulcer/stomach cancer), Listeria monocytogenes (listeriosis), Staphylococcus aureus (major cause of nosocomial infection), S. pyogenes (major cause of nosocomial infections) and S. pneumoniae (pneumonia). Dark magenta, 100% conserved; tan, 63%; dark cyan, 25%. Alignments were constructed with CLUSTALW2 and molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081) (Pettersen, et al., 2004). Supported by Figure S3.

Figure 3. C91 inhibits H. pylori growth

a. Compound C91 in DMSO was added to freshly diluted stationary cultures of H. pylori strain G27 in Brucella broth. Samples were removed at the indicated time points, diluted, and plated to determine bacterial proliferation/survival. Each point is the average of duplicate determinations; a representative of three experiments is shown. Black, DMSO alone. C91 concentrations: purple, 2 µM; blue, 7 µM; green, 20 µM; orange, 60 µM; red, 200 µM. b. Compound C91 was added to freshly diluted cultures of E. coli MG1655 in Luria broth. Each point is the average of three determinations; the standard deviations are smaller than the point. Black, DMSO alone. C91 concentrations: orange, 100 µM; red, 200 µM. Supported by Figure S4.