Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.     

Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized Reactive Red 120

Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).     

Purine salvage enzymes of parasites as targets for structure-based inhibitor design

Nearly 30 years have passed since purine salvage enzymes were first proposed as targets of drugs in the chemotherapeutic treatment of diseases caused by parasites. The rationale behind a structure-based approach to the design of chemotherapeutic agents involves the use of information about substrate preference and the three-dimensional structure of a target enzyme to design potent selective inhibitors of that enzyme. This approach is outlined here by Syd Craig and Ann Eakin, as it applies to the possible design of inhibitors of a purine salvage enzyme, the hypoxanthine phosphoribosyltransferase.     

Structural bioinformatics study of PNP from Schistosoma mansoni

The parasite Schistosoma mansoni lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. Schistosomiasis is endemic in 76 countries and territories and amongst the parasitic diseases ranks second after malaria in terms of social and economic impact and public health importance. The PNP is an attractive target for drug design and it has been submitted to extensive structure-based design. The atomic coordinates of the complex of human PNP with inosine were used as template for starting the modeling of PNP from S. mansoni complexed with inosine. Here we describe the model for the complex SmPNP-inosine and correlate the structure with differences in the affinity for inosine presented by human and S. mansoni PNPs.     

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.     

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.     

Trifluoromethylpyrimidine-based inhibitors of proline-rich tyrosine kinase 2 (PYK2): structure-activity relationships and strategies for the elimination of reactive metabolite formation

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.     

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 μM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.     

Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc

The structure–activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities. The improved inhibitors possessed enhanced selectivity profiles (rationalized using the X-ray crystallographic data) against other nuclear receptors.     

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.     

Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.     

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.     

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.     

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.     

Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 A resolution

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.     

Identification and characterization of small molecule inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum causes the most deadly form of malaria and accounts for over one million deaths annually. The malaria parasite is unable to salvage pyrimidines and relies on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHOD), a mitochondrially localized flavoenzyme, catalyzes the rate-limiting step of this pathway and is therefore an attractive antimalarial chemotherapeutic target. Using a target-based high throughput screen, we have identified a series of potent, species-specific inhibitors of P. falciparum DHOD (pfDHOD) that are also efficacious against three cultured strains (3D7, HB3, and Dd2) of P. falciparum. The primary antimalarial mechanism of action of these compounds was confirmed to be inhibition of pfDHOD through a secondary assay with transgenic malaria parasites, and the structural basis for enzyme inhibition was explored through in silico structure-based docking and site-directed mutagenesis. Compound-mediated cytotoxicity was not observed with human dermal fibroblasts or renal epithelial cells. These data validate pfDHOD as an antimalarial drug target and provide chemical scaffolds with which to begin medicinal chemistry efforts.     

Design and synthesis of novel proline based factor XIa selective inhibitors as leads for potential new anticoagulants

A series of 4, 4-disubstituted proline analogs were designed, synthesized, and tested for selective inhibition of blood coagulation factor XIa in search of new non-vitamin K antagonists based oral anticoagulants for potential prevention and treatment of thrombotic diseases. Starting from a potent thrombin (FIIa) inhibitor chemotype with FIIa IC₅₀ = 1 nM and FXIa IC₅₀ = 160 nM, medicinal chemistry iterations guided by molecular modeling and structure-based drug design led to steady improvement of FXIa potency while dialing down thrombin activity and improving selectivity. Through this exercise, a thousand-fold enhancement of selectivity over thrombin was achieved with some analogs carrying factor XIa inhibition potencies in the 10 nM range. In this communication, we discuss the design principles and structure activity relationship (SAR) of these novel FXIa selective inhibitors.     

Tetrabutylammonium fluoride-assisted rapid N9-alkylation on purine ring: application to combinatorial reactions in microtiter plates for the discovery of potent sulfotransferase inhibitors in situ

Tremendous efforts have been invested in the synthesis of purine libraries due to their importance in targeting various enzymes involved in different diseases and cellular processes. The synthesis of N9-alkylated purine scaffolds relied mostly on Mitsunobu conditions with a variety of alcohols or strong basic conditions with different organic halides. A more reliable and efficient way for the synthesis of N(9)-alkylated purine scaffolds is reported. This method uses tetrabutylammonium fluoride (TBAF) to assist such chemistry. In many cases, the reactions were completed within 10 min and gave the desired product in high yield and selectivity. Moreover, these mild reaction conditions permitted its use in combinatorial reactions in microtiter plates followed by in situ screening for the discovery of potent sulfotransferase inhibitors.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.