Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

• MeSH
• Enzyme Inhibitors * pharmacology   chemistry   chemical synthesis   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Pentosyltransferases * antagonists & inhibitors   metabolism   MeSH
• Drug Design * MeSH
• Trypanosoma brucei brucei drug effects   enzymology   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Browser
• Enzyme Inhibitors * MeSH
• Pentosyltransferases * MeSH

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

• MeSH
• Adenine Phosphoribosyltransferase metabolism   MeSH
• Adenine Nucleotides metabolism   MeSH
• Cell Line MeSH
• HeLa Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nucleosides metabolism   MeSH
• Organophosphonates metabolism   MeSH
• Purines metabolism   MeSH
• Trypanosoma brucei brucei metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine Phosphoribosyltransferase MeSH
• Adenine Nucleotides MeSH
• Nucleosides MeSH
• Organophosphonates MeSH
• purine MeSH Browser
• Purines MeSH

Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed.IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the ΔhgprtM. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

• Keywords
• Mycobacterium smegmatis, guanine, hypoxanthine, hypoxanthine-guanine phosphoribosyltransferase, inhibitors, purine salvage pathway,
• MeSH
• Antitubercular Agents chemistry   pharmacology   MeSH
• Hypoxanthine Phosphoribosyltransferase antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Enzyme Inhibitors chemistry   pharmacology   MeSH
• Catalysis MeSH
• Metabolic Networks and Pathways MeSH
• Microbial Viability MeSH
• Mycobacterium smegmatis genetics   growth & development   metabolism   MeSH
• Plasmids genetics   MeSH
• Purines metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antitubercular Agents MeSH
• Hypoxanthine Phosphoribosyltransferase MeSH
• Enzyme Inhibitors MeSH
• purine MeSH Browser
• Purines MeSH

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

• MeSH
• Hypoxanthine Phosphoribosyltransferase antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Enzyme Inhibitors chemistry   pharmacology   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Metabolic Networks and Pathways drug effects   MeSH
• Models, Molecular MeSH
• Drug Discovery MeSH
• Pentosyltransferases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Purinones metabolism   MeSH
• RNA Interference MeSH
• Trypanocidal Agents chemistry   pharmacology   MeSH
• Trypanosoma brucei brucei drug effects   enzymology   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Browser
• Hypoxanthine Phosphoribosyltransferase MeSH
• Enzyme Inhibitors MeSH
• Pentosyltransferases MeSH
• Purinones MeSH
• Trypanocidal Agents MeSH