Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability.

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.

Synthesis and evaluation of symmetric acyclic nucleoside bisphosphonates as inhibitors of the Plasmodium falciparum, Plasmodium vivax and human 6-oxopurine phosphoribosyltransferases and the antimalarial activity of their prodrugs.

Two new series of symmetric acyclic nucleoside bisphosphonates (ANbPs) have been synthesised as potential inhibitors of the Plasmodium falciparum (Pf) and vivax (Pv) 6-oxopurine phosphoribosyltransferases. The structural variability between these symmetric ANbPs lies in the number of atoms in the two acyclic linkers connecting the N9 atom of the purine base to each of two phosphonate groups and the branching point of the acyclic moiety relative to the purine base, which occurs at either the alpha or beta positions. Within each series, six different 6-oxopurine bases have been attached. In general, the ANbPs with either guanine or hypoxanthine have lower Ki values than for those containing either the 8-bromo or 7-deaza 6-oxopurine bases. The lowest Ki values obtained for the two parasite enzymes were 0.1µM (Pf) and 0.2µM (Pv) for this series of compounds. Two phosphoramidate prodrugs of these inhibitors exhibited antimalarial activity against Pf in infected erythrocyte cell culture with IC50 values of 0.8 and 1.5µM. These two compounds exhibited low cytotoxicity in human A549 cells having CC50 values of >300µM resulting in an excellent selectivity index.

N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides.

Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.

Antimalarial activity of prodrugs of N-branched acyclic nucleoside phosphonate inhibitors of 6-oxopurine phosphoribosyltransferases.

Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases (PRTs), key enzymes of the purine salvage pathway. Chemical modifications, based on the crystal structures of several inhibitors in complex with the human PRTase, led to the design of a new class of inhibitors--the aza-ANPs. Because of the negative charges of the phosphonic acid moiety, their ability to cross cell membranes is, however, limited. Thus, phosphoramidate prodrugs of the aza-ANPs were prepared to improve permeability. These prodrugs arrest parasitemia with IC50 values in the micromolar range against Plasmodium falciparum-infected erythrocyte cultures (both chloroquine-sensitive and chloroquine-resistant Pf strains). The prodrugs exhibit low cytotoxicity in several human cell lines. Thus, they fulfill two essential criteria to qualify them as promising antimalarial drug leads.

MYC Induces Oncogenic Stress through RNA Decay and Ribonucleotide Catabolism in Breast Cancer.

Upregulation of MYC is a hallmark of cancer, wherein MYC drives oncogenic gene expression and elevates total RNA synthesis across cancer cell transcriptomes. Although this transcriptional anabolism fuels cancer growth and survival, the consequences and metabolic stresses induced by excess cellular RNA are poorly understood. Herein, we discover that RNA degradation and downstream ribonucleotide catabolism is a novel mechanism of MYC-induced cancer cell death. Combining genetics and metabolomics, we find that MYC increases RNA decay through the cytoplasmic exosome, resulting in the accumulation of cytotoxic RNA catabolites and reactive oxygen species. Notably, tumor-derived exosome mutations abrogate MYC-induced cell death, suggesting excess RNA decay may be toxic to human cancers. In agreement, purine salvage acts as a compensatory pathway that mitigates MYC-induced ribonucleotide catabolism, and inhibitors of purine salvage impair MYC+ tumor progression. Together, these data suggest that MYC-induced RNA decay is an oncogenic stress that can be exploited therapeutically. Significance: MYC is the most common oncogenic driver of poor-prognosis cancers but has been recalcitrant to therapeutic inhibition. We discovered a new vulnerability in MYC+ cancer where MYC induces cell death through excess RNA decay. Therapeutics that exacerbate downstream ribonucleotide catabolism provide a therapeutically tractable approach to TNBC (Triple-negative Breast Cancer) and other MYC-driven cancers.

N2'-Branched Acyclic Nucleoside Phosphonates Containing 9-Deazahypoxanthine as Inhibitors of Plasmodium falciparum 6-Oxopurine Phosphoribosyltransferase.

Twelve N2'-branched acyclic nucleoside phosphonates and bisphosphonates were synthesized as potential inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), the key enzyme in the purine salvage pathway for production of purine nucleotides. The chemical structures were designed with the aim to study selectivity of the inhibitors for PfHGXPRT over human HGPRT. The newly prepared compounds contain 9-deazahypoxanthine connected to a phosphonate group via a five-atom-linker bearing a nitrogen atom (N2') as a branching point. All compounds, with the additional phosphonate group(s) in the second aliphatic linker attached to N2' atom, were low micromolar inhibitors of PfHGXPRT with low to modest selectivity for the parasite enzyme over human HGPRT. The effect of the addition of different chemical groups/linkers to N2' atom on the inhibition constants and selectivity is discussed.

Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents.

6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2'-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-α-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2µM and 5µM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.

Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases.

The purine salvage enzyme, hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT], catalyses the synthesis of the purine nucleoside monophosphates, IMP, GMP or XMP essential for DNA/RNA production. In protozoan parasites, such as Plasmodium, this is the only route available for their synthesis as they lack the de novo pathway which is present in human cells. Acyclic nucleoside phosphonates (ANPs), analogs of the purine nucleoside monophosphates, have been found to inhibit Plasmodium falciparum (Pf) HGXPRT and Plasmodium vivax (Pv) HGPRT with K(i) values as low as 100 nM. They arrest parasitemia in cell based assays with IC(50) values of the order of 1-10 µM. ANPs with phosphonoalkyl and phosphonoalkoxyalkyl moieties linking the purine base and phosphonate group were designed and synthesised to evaluate the influence of this linker on the potency and/or selectivity of the ANPs for the human and malarial enzymes. This data shows that variability in the linker, as well as the positioning of the oxygen in this linker, influences binding. The human enzyme binds the ANPs with K(i) values of 0.5 µM when the number of atoms in the linker was 5 or 6 atoms. However, the parasite enzymes have little affinity for such long chains unless oxygen is included in the three-position. In comparison, all three enzymes have little affinity for ANPs where the number of atoms linking the base and the phosphonate group is of the order of 2-3 atoms. The chemical nature of the purine base also effects the K(i) values. This data shows that both the linker and the purine base play an important role in the binding of the ANPs to these three enzymes.

Synthesis and anti-trypanosomal evaluation of novel N-branched acyclic nucleoside phosphonates bearing 7-aryl-7-deazapurine nucleobase.

A series of novel 7-aryl-7-deazaadenine-based N-branched acyclic nucleoside phosphonates (aza-ANPs) has been prepared using the optimized Suzuki cross-coupling reaction as the key synthetic step. The final free phosphonates 15a-h were inactive, due to their inefficient transport across cell membranes, but they inhibited Trypanosoma brucei adenine phosphoribosyltransferase (TbrAPRT1) with Ki values of 1.7-14.1 µM. The corresponding phosphonodiamidate prodrugs 14a-h exhibited anti-trypanosomal activity in the Trypanosoma brucei brucei cell-based assay with EC50 values in the range of 0.58-6.8 µM. 7-(4-Methoxy)phenyl-7-deazapurine derivative 14h, containing two phosphonate moieties, was the most potent anti-trypanosomal agent from the series, with EC50 = 0.58 µM and SI = 16. Finally, phosphonodiamidate prodrugs 14a-h exerted low micromolar cytotoxicity against leukemia and/or cancer cell lines tested.

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases.

Pathogens such as Plasmodium and Trypanosoma spp. are unable to synthesize purine nucleobases. They rely on the salvage of these purines and their nucleosides from the host cell to synthesize the purine nucleotides required for DNA/RNA production. The key enzymes in this pathway are purine phosphoribosyltransferases (PRTs). Here, we synthesized 16 novel acyclic nucleoside phosphonates, 12 with a chiral center at C-2', and eight bearing a second chiral center at C-6'. Of these, bisphosphonate (S,S)-48 is the most potent inhibitor of the Plasmodium falciparum and P. vivax 6-oxopurine PRTs and the most potent inhibitor of two Trypanosoma brucei (Tbr) 6-oxopurine PRTs yet discovered, with Ki values as low as 2 nM. Crystal structures of (S,S)-48 in complex with human and Tbr 6-oxopurine PRTs show that the inhibitor binds to the enzymes in different conformations, providing an explanation for its potency and selectivity (i.e., 35-fold in favor of the parasite enzymes).

Acyclic nucleoside phosphonates with a branched 2-(2-phosphonoethoxy)ethyl chain: efficient synthesis and antiviral activity.

Series of novel acyclic nucleoside phosphonates (ANPs) with various nucleobases and 2-(2-phosphonoethoxy)ethyl (PEE) chain bearing various substituents in ß-position to the phosphonate moiety were prepared. The influence of structural alternations on antiviral activity was studied. Several derivatives exhibit antiviral activity against HIV and vaccinia virus (middle micromolar range), HSV-1 and HSV-2 (lower micromolar range) and VZV and CMV (nanomolar range), although the parent unbranched PEE-ANPs are inactive. Adenine as a nucleobase and the methyl group attached to the PEE chain proved to be a prerequisite to afford pronounced antiviral activity.

Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro.

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).

Nucleotide analogues containing a pyrrolidine, piperidine or piperazine ring: Synthesis and evaluation of inhibition of plasmodial and human 6-oxopurine phosphoribosyltransferases and in vitro antimalarial activity.

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 µM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5-12.1 µM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 µM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 µM suggesting good selectivity for further structure-activity relationship investigations.

C1'-Branched acyclic nucleoside phosphonates mimicking adenosine monophosphate: Potent inhibitors of Trypanosoma brucei adenine phosphoribosyltransferase.

Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered. Herein, we designed and synthesized two series of new ANPs branched at the C1' position as mimics of adenosine monophosphate. The novel ANPs efficaciously inhibited Trypanosoma brucei adenine PRT (TbrAPRT1) activity in vitro and it was shown that the configuration on the C1' chiral centre strongly influenced their activity: the (R)-enantiomers proved to be more potent compared to the (S)-enantiomers. Two ANPs, with Ki values of 0.39 µM and 0.57 µM, represent the most potent TbrAPRT1 inhibitors reported to date and they are an important tool to further study purine metabolism in various parasites.

Helicobacter pylori Xanthine-Guanine-Hypoxanthine Phosphoribosyltransferase-A Putative Target for Drug Discovery against Gastrointestinal Tract Infections.

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the salvage enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a K(i) of 1 microM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.

Crystal structures of Trypanosoma brucei hypoxanthine - guanine - xanthine phosphoribosyltransferase in complex with IMP, GMP and XMP.

The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.

Sulfide, sulfoxide and sulfone bridged acyclic nucleoside phosphonates as inhibitors of the Plasmodium falciparum and human 6-oxopurine phosphoribosyltransferases: Synthesis and evaluation.

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a recognized target for antimalarial chemotherapeutics. It synthesises all of the 6-oxopurine nucleoside monophosphates, IMP, GMP and XMP needed by the malarial parasite, Plasmodium falciparum (Pf). PfHGXPRT is also indirectly responsible for the synthesis of the adenosine monophosphate, AMP. The acyclic nucleoside phosphonates (ANPs) are a class of PfHGXPRT inhibitors. Prodrugs of these compounds are able to arrest the growth of Pf in cell culture. In the search for new inhibitors of PfHGXPRT, a series of sulfur containing ANPs (thia-ANPs) has been designed and synthesized. These compounds are based on the structure of 2-(phosphonoethoxy)ethylguanine (PEEG) and PEEHx which consist of a purine base (i.e. guanine or hypoxanthine) linked to a phosphonate group by five atoms i.e. four carbons and one oxygen. Here, PEEG and PEEHx were modified by substituting a sulfide, sulfoxide or a sulfone bridge for the oxygen atom in the linker. The effect of these substitutions on the Ki values for human HGPRT and PfHGXPRT was investigated and showed that most of the thia-ANPs distinctively favour PfHGXPRT. For example, the thia-analogue of PEEHx has a Ki value of 0.2 µM for PfHGXPRT, a value 25-fold lower than for the human counterpart. Prodrugs of these compounds have IC50 values in the 4-6 µM range in antimalarial cell-based assays, making them attractive compounds for further development as antimalarial drug leads.

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

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.

Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics.

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has Ki values of 8 and 7 nM, respectively, for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 Å resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl l-phenylalanine) tetraamide prodrug of this compound was synthesized, and it has an IC50 of 11.7 ± 3.2 µM against Pf lines grown in culture and a CC50 in human A549 cell lines of 102 ± 11 µM, thus giving it a ∼10-fold selectivity index.