Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

Characterization of Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Exploring Subsite Selectivity within Plasmodium vivax N-Myristoyltransferase Using Pyrazole-Derived Inhibitors.

N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.

Selective Bias Virtual Screening for Discovery of Promising Antimalarial Candidates targeting Plasmodium N-Myristoyltransferase.

Malaria remains a significant public health challenge, with Plasmodium vivax being the species responsible for the most prevalent form of the disease. Given the limited therapeutic options available, the search for new antimalarials against P. vivax is urgent. This study aims to identify new inhibitors for P. vivax N-myristoyltransferase (PvNMT), an essential drug target against malaria. Through a validated virtual screening campaign, we prioritized 23 candidates for further testing. In the yeast NMT system, seven compounds exhibit a potential inhibitor phenotype. In vitro antimalarial phenotypic assays confirmed the activity of four candidates while demonstrating an absence of cytotoxicity. Enzymatic assays reveal LabMol-394 as the most promising inhibitor, displaying selectivity against the parasite and a strong correlation within the yeast system. Furthermore, molecular dynamics simulations shed some light into its binding mode. This study constitutes a substantial contribution to the exploration of a selective quinoline scaffold and provides valuable insights into the development of new antimalarial candidates.

Identification of potent and selective N-myristoyltransferase inhibitors of Plasmodium vivax liver stage hypnozoites and schizonts.

Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage.

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

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 counter-screened 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.

Identification of P218 as a potent inhibitor of Mycobacterium ulcerans DHFR.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a debilitating chronic disease that mainly affects the skin. Current treatments for Buruli ulcer are efficacious, but rely on the use of antibiotics with severe side effects. The enzyme dihydrofolate reductase (DHFR) plays a critical role in the de novo biosynthesis of folate species and is a validated target for several antimicrobials. Here we describe the biochemical and structural characterization of M. ulcerans DHFR and identified P218, a safe antifolate compound in clinical evaluation for malaria, as a potent inhibitor of this enzyme. We expect our results to advance M. ulcerans DHFR as a target for future structure-based drug discovery campaigns.