Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.

Structure-based inhibitor design.

Time and costs associated with the discovery of new drugs have been significantly reduced by enzyme structure-based approaches to the discovery of new chemotherapeutic agents. However, fundamental components of the overall approach continue to rely on technologies which, by their nature, involve relatively random processes (i.e., combinatorial chemistry and high-throughput screening). Thus, the efficiency of the drug discovery process potentially could be further improved through better use of structural information. In this regard, three-dimensional structures of enzymes are now being solved at high resolution and/or in conformations that provide data that should be more useful for inhibitor design or discovery. Scientists are beginning to appreciate the importance of water as a possible competitor of inhibitors for binding to target enzymes. New computational algorithms are improving the efficiency of identifying flexible inhibitors from among the large numbers of compounds in chemical databases. Also, tools of molecular genetics together with structures of target enzymes are likely to be used more frequently in dealing with the development of resistance to novel chemotherapeutic agents. Instead of detailing success stories in structure-based drug discovery, the following article considers how future efforts to discover or design new drugs may increasingly rely on information about molecular targets and less on data acquired via approaches involving random methodologies.

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Limited proteolysis of a trypanosomal hypoxanthine phosphoribosyltransferase yields crystals that diffract X-rays to near atomic resolution.

Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.

Approaching the transition state in the crystal structure of a phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferase (HPRT) salvages 6-oxopurine bases in the nucleotide metabolic pathway. The 1.8 A crystal structure of an asymmetric dimer of the HPRT from the protozoan parasite Trypanosoma cruzi was determined in a ternary complex with the primary substrate phosphoribosylpyrophosphate (PRPP) and an analogue of the substrate hypoxanthine, revealing both open and closed active site conformations. The ligands are positioned for in-line nucleophilic attack at the PRPP ribose C1' by two metal ions which straddle the pyrophosphate leaving group. The structure provides the first evidence for the involvement of two metal ions in the HPRT-catalyzed reaction, and structural details further suggest the mechanism may proceed via SN2-type chemistry. The closed conformation reveals the structural roles for invariant flexible loop residues Ser103 and Tyr104 and supports a role for the loop in the liberation of pyrophosphate. The pre-transition state structure is valuable for understanding the enzyme mechanism, as well as providing a foundation for antiparasite drug design efforts against T. cruzi, which causes Chagas' disease in humans. Additionally, the structure illuminates the molecular basis of three inherited mutations in the human HPRT leading to Lesch-Nyhan syndrome (D193N) or gout (S103R or S109L), as the homologous residues in the trypanosomal enzyme contribute to the previously unrecognized Mg2+ ion binding site and to the formation of the closed flexible loop, respectively.

A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.

Bacterial complementation as a means to test enzyme-ligand interactions.

A bacterial complementation assay has been developed for the rapid screening of a large number of compounds to identify those that inhibit an enzyme target for structure-based inhibitor design. The target enzyme is the hypoxanthine phosphoribosyltransferase (HPRT). This enzyme has been proposed as a potential target for inhibitors that may be developed into drugs for the treatment of diseases caused by several parasites. The screening assay utilizes genetically deficient bacteria complemented by active, recombinant enzyme grown in selective medium in microtiter plates. By comparing absorbance measurements of bacteria grown in the presence and absence of test compounds, the effect of the compounds on bacterial growth can be rapidly assayed. IC50 values for inhibition of bacterial growth are a reflection of the ability of the compounds to bind and/or inhibit the recombinant enzyme. We have tested this bacterial complementation screening assay using recombinant HPRT from the parasites. Plasmodium falciparum and Trypanosoma cruzi, as well as the human enzyme. The results of these studies demonstrate that a screening assay using bacterial complement selection can be used to identify compounds that target enzymes and can become an important part of structure-based drug design efforts.

A single amino acid substitution in the human and a bacterial hypoxanthine phosphoribosyltransferase modulates specificity for the binding of guanine.

Early studies involving purine salvage in Salmonella typhimurium resulted in the isolation and identification of a mutant strain possessing a genetically modified hypoxanthine phosphoribosyl-transferase (HPRT) with enhanced substrate specificity for guanine [Benson, C. E., and Gots, J. S. (1975) J. Bacteriol. 121, 77-82]. To explore the molecular basis for this altered substrate specificity in the mutant hpt gene product, degenerate oligonucleotide primers, designed according to the N- and C-termini of the HPRT of Escherichia coli, were used in polymerase chain reactions to amplify both the mutant and wild-type S. typhimurium hpt genes from genomic DNA. Analysis of the deduced amino acid sequences revealed that a single base mutation resulted in the encoding of a Thr in the mutant HPRT, instead of an Ile found in the wild-type enzyme, at a position analogous to position 192 (Leu-192) of the human HPRT. Comparison of kinetic data for purified recombinant mutant and wild-type HPRTs showed no difference in the overall catalytic efficiency (kcat/K(m)) with hypoxanthine as substrate, but with guanine, the mutant enzyme exhibited a more than 50-fold higher kcat/K(m) largely as a result of a decrease of nearly 2 orders of magnitude in K(m). Involvement in substrate binding of the cognate amino acid at position 192 in the human HPRT was investigated using site-directed mutagenesis. Mutation of Leu-192 to Thr did not significantly alter kcat/K(m) values for hypoxanthine and guanine compared to wild-type, and replacement of Leu-192 with Ile had no significant change in kinetics for either hypoxanthine or PRPP. However, this Ile substitution resulted in an over 15-fold decrease in the kcat/K(m) for guanine due to a greater than 15-fold increase in K(m). These results demonstrate that a single active site amino acid substitution in HPRTs can significantly alter the specificity for binding guanine.

Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi is a potential target for enzyme structure-based inhibitor design, based on previous studies which indicate that these parasites lack the metabolic enzymes required for de novo synthesis of purine nucleotides. By using a bacterial complement selection system, 59 purine analogs were assayed for their interaction with the HPRTs from T. cruzi and Homo sapiens. Eight compounds were identified from the bacterial assay to have an affinity for the trypanosomal enzyme. Inhibition constants for four of these compounds against purified recombinant trypanosomal and human HPRTs were determined and compared. The results confirm that the recombinant system can be used to identify compounds which have affinity for the trypanosomal HPRT. Furthermore, the results provide evidence for the importance of chemical modifications at positions 6 and 8 of the purine ring in the binding of these compounds to the HPRTs. An accurate three-dimensional structure of the trypanosomal enzyme will greatly enhance our understanding of the interactions between HPRTs and these compounds. Toward this end, crystallization conditions for the trypanosomal HPRT and preliminary analysis of X-ray diffraction data to a resolution of 2 A is reported. These results represent significant progress toward a structure-based approach to the design of inhibitors of the HPRT of trypanosomes with the long-range goal of developing new drugs for the treatment of Chagas' disease.

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.

Substitution of lysine for arginine at position 199 of a hypoxanthine phosphoribosyltransferase interferes with binding of the primary substrate to the active site.

Lysine was substituted for a conserved arginine at position 199 of the schistosomal hypoxanthine phosphoribosyltransferase (HPRT). This resulted in a > or = 35-fold increase in the K(M) for binding phosphoribosyl-pyrophosphate (PRPP). The possible functional role of R199 in tertiary structure, as well as in the binding of PRPP, is interpreted in the context of the reported three dimensional structure for the human HPRT.

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites.

Expression plasmids encoding the hypoxanthine phosphoribosyltransferases (HPRTs) of Plasmodium falciparum, Schistosoma mansoni, Tritrichomonas foetus, and Homo sapiens were subcloned into genetically deficient Escherichia coli that requires complementation by the activity of a recombinant HPRT for growth on semidefined medium. Fifty-nine purine analogs were screened for their abilities to inhibit the growth of these bacteria. Several compounds that selectively altered the growth of the bacteria complemented by the malarial, schistosomal, or tritrichomonal HPRT compared with the growth of bacteria expressing the human enzyme were identified. These results demonstrate that the recombinant approach to screening compounds by complement selection in a comparative manner provides a rapid and efficient method for the identification of new lead compounds selectively targeted to the purine salvage enzymes of parasites.

Differential inhibitory effects of GMP-2',3'-dialdehyde on human and schistosomal hypoxanthine-guanine phosphoribosyltransferases.

The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of human and the parasitic trematode, Schistosoma mansoni, were expressed at high levels in transformed Escherichia coli in their native forms. Guanosine 2',3'-dialdehyde 5'-phosphate (ox-GMP) was shown to bind irreversibly to both enzymes in a time-dependent manner. This binding was stabilized by sodium borohydride reduction, suggesting that a Schiff's base is formed between the dialdehyde groups of ox-GMP and the amino group of a lysine residue in the enzymes. This linkage formation applies also to inosine 2',3'-dialdehyde 5'-phosphate but not to adenosine 2',3'-dialdehyde 5'-phosphate. GMP was found to be protective against ox-GMP inactivation and [3H]ox-GMP labeling of both HGPRTases. 5-Phosphoribosyl-1-diphosphate (PRibPP) also protects human HGPRTase against the ox-GMP inactivation and [3H]ox-GMP labeling but provides virtually no protection against the ox-GMP inactivation and labeling of the schistosomal enzyme, even though PRibPP binds to the latter with a threefold higher affinity. These results imply that PRibPP and ox-GMP compete with each other for binding to the human HGPRTase but not for binding to the schistosomal enzyme. This discrepancy could be exploited for the purpose of designing selective inhibitors of the schistosomal HGPRTase. Guanosine 2',3'-dialdehyde (ox-guanosine) is nearly as active as ox-GMP in inhibiting schistosomal HGPRTase but much less potent in inhibiting human HGPRTase, suggesting that ox-guanosine and ox-GMP may bind equally well to the parasite enzyme. PRibPP can protect human but not schistosomal HGPRTase against the inactivation by ox-guanosine. Therefore, ox-GMP and ox-guanosine must be forming Schiff's bases with the same amino acid residues in each of the two HGPRTases.

Sequence of human protein serine/threonine phosphatase 1 gamma and localization of the gene (PPP1CC) encoding it to chromosome bands 12q24.1-q24.2.

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, termed PP1 gamma, was isolated from a human teratocarcinoma library. The sequence suggests that alternative splicing produces two forms of PP1 gamma, designated PP1 gamma 1 and PP1 gamma 2, which differ in their C-termini. The gene for human PP1 gamma (PPP1CC) was localized to chromosome 12 by analysis of somatic cell hybrid DNA and mapped to bands q24.1-q24.2 by in situ hybridisation. These data show that although PP1 gamma 1 and PP1 gamma 2 are 94% and 93% identical to PP1 alpha respectively, the PP1 gamma gene is not closely linked to the PP1 alpha gene, which has been mapped to chromosome 11.

Comparing the human and schistosomal hypoxanthine-guanine phosphoribosyltransferases by circular dichroism.

The hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) of human and the parasitic trematode, Schistosoma mansoni, are of biomedical importance. The conformations of these two enzymes were studied by circular dichroism (CD). The schistosomal HGPRTase is estimated to contain 27% alpha-helix and 30% beta-structure. This result is consistent with what is predicted from a tertiary model (Craig, S.P., Cohen, F.E., Yuan, L., McKerrow, J.H. and Wang, C.C. (1991) in Molecular & Immunological Aspects of Parasitism (Wang, C.C., ed.), pp. 122-138, Am. Assoc. Adv. Sci., Washington DC, USA), which proposes that the enzyme is an alpha/beta barrel protein. The human enzyme is estimated to contain 21% alpha-helix and 53% beta-form. The two enzymes are different in their thermostability. The human enzyme remains active after being heated to 85 degrees C for 15 min, while the schistosomal enzyme only retains its activity at temperature below 65 degrees C. The transition temperature (T1/2) of the schistosomal HGPRTase was determined by CD measurement to be 57.5 degrees C. One of the enzyme substrates, phosphoribose pyrophosphate (PRPP), stabilizes the HGPRTases by preventing the human enzyme from unfolding at 85 degrees C and partially protecting the schistosomal enzyme from unfolding at 65 degrees C. It is suggested that the amino-acid substitutions in the human enzyme improve the spatial structure and stability of its alpha-helices, which may lead to an enhanced thermostability.

The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters.

Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5'-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.

Localisation of the gene for human aromatic L-amino acid decarboxylase (DDC) to chromosome 7p13-->p11 by in situ hybridisation.

The human gene for aromatic L-amino acid decarboxylase (DDC) was previously assigned to chromosome 7 by analysis of a panel of somatic cell hybrids. We report here refinement of this localisation, by in situ hybridisation, to 7p13-->p11.

Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase.

Schistosomiasis is a trematode infection of some 200 million people. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy [Dovey, H. F., McKerrow, J. H., & Wang, C. C. (1984) Mol. Biochem. Parasitol, 11, 157-167]. The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions. Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions. In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine. Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP. Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi. The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS)