AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma.

BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. METHODS: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. RESULTS: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. CONCLUSIONS: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.

Patterns of interaction between genetic and nongenetic attributes and methotrexate efficacy in rheumatoid arthritis.

OBJECTIVE: The contribution of low-penetrance single nucleotide polymorphisms to methotrexate efficacy in rheumatoid arthritis (RA) is inconsistent between studies. We sought to elucidate architecture of methotrexate response in three cohorts of patients with RA treated with methotrexate. METHODS: Single nucleotide polymorphism frequencies in genes from folate, purine, and pyrimidine pathways were measured to develop a model of gene-gene interactions using multifactor dimensionality reduction in 439 patients who received methotrexate in the USA and The Netherlands. A third cohort of 530 patients with RA from Sweden was used to replicate the findings. Methotrexate efficacy was assessed using the European League Against Rheumatism criteria in the majority of patients. RESULTS: Nonlinear patterns of gene-gene interactions between variants in aminoimidazole carboxamide ribonucleotide transformylase (C347G), reduced-folate carrier (G80A) and inosine-triphosphate pyrophosphatase (C94A) revealed a predisposing genetic attribute significantly associated with methotrexate response in the USA and Dutch cohorts [odds ratio (OR)=2.9, 95% confidence interval (CI): 1.9-4.2; P<0.001]. Although the finding was not replicated in the Swedish cohort (OR=0.9; 95% CI: 0.64-1.37; P=0.74) a multifactor dimensionality reduction analysis superimposing the predisposing genetic attribute with patient's age, sex, and anticitrullinated peptide antibodies positivity (ACPA) revealed a pattern of interaction significant in all three cohorts (OR=2.2, 95% CI: 1.6-2.9; P<0.01). The selective advantage toward response in the presence of the predisposing genetic attribute was lost in females and ACPA-positive patients, whereas older and male ACPA-negative patients tended to exhibit a greater likelihood of response in the absence of the predisposing genetic attribute. CONCLUSION: Gene-gene interactions together with nongenetic attributes may contribute to methotrexate efficacy in RA.

Expression, purification, crystallization and preliminary X-ray diffraction crystallographic study of PurH from Escherichia coli.

In bacteria and eukaryotes, the last two steps of de novo purine biosynthesis are catalyzed by bifunctional purine-biosynthesis protein (PurH), which is composed of two functionally independent domains linked by a flexible region. The N-terminal domain possesses IMP cyclohydrolase activity and the C-terminal domain possesses aminoimidazole-4-carboxamide ribonucleotide transformylase activity. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of PurH from Escherichia coli with an N-terminal His(6) tag. The crystals diffracted to a maximum resolution of 3.05 Å and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 76.37, b = 132.15, c = 82.64 Å, ß = 111.86°.

Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria.

The class Gammaproteobacteria, which forms one of the largest groups within bacteria, is currently distinguished from other bacteria solely on the basis of its branching in phylogenetic trees. No molecular or biochemical characteristic is known that is unique to the class Gammaproteobacteria or its different subgroups (orders). The relationship among different orders of gammaproteobacteria is also not clear. In this study, we present detailed phylogenomic and comparative genomic analyses on gammaproteobacteria that clarify some of these issues. Phylogenetic trees based on concatenated sequences for 13 and 36 universally distributed proteins were constructed for 45 members of the class Gammaproteobacteria covering 13 of its 14 orders. In these trees, species from a number of the subgroups formed distinct clades and their relative branching order was indicated as follows (from the most recent to the earliest diverging): Enterobacteriales >Pasteurellales >Vibrionales, Aeromonadales >Alteromonadales >Oceanospirillales, Pseudomonadales >Chromatiales, Legionellales, Methylococcales, Xanthomonadales, Cardiobacteriales, Thiotrichales. Four conserved indels in four widely distributed proteins that are specific for gammaproteobacteria are also described. A 2 aa deletion in 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase (AICAR transformylase; PurH) was a distinctive characteristic of all gammaproteobacteria (except Francisella tularensis). Two other conserved indels (a 4 aa deletion in RNA polymerase beta-subunit and a 1 aa deletion in ribosomal protein L16) were found uniquely in various species of the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales, but were not found in other gammaproteobacteria. Lastly, a 2 aa deletion in leucyl-tRNA synthetase was commonly present in the above orders of the class Gammaproteobacteria and also in some members of the order Oceanospirillales. The presence of the conserved indels in these gammaproteobacterial orders indicates that species from these orders shared a common ancestor that was separate from other bacteria, a suggestion that is supported by phylogenetic studies. Systematic blastp searches were also conducted on various open reading frames (ORFs) in the genome of Escherichia coli K-12. These analyses identified 75 proteins that were unique to most members of the class Gammaproteobacteria or were restricted to species from some of its main orders (Enterobacteriales; Enterobacteriales and Pasteurellales; Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales; and the Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales, Alteromonadales, Oceanospirillales and Pseudomonadales etc.). The genes for these proteins have evolved at various stages during the evolution of gammaproteobacteria and their species distribution pattern, in conjunction with other results presented here, provide valuable information regarding the evolutionary relationships among these bacteria.

The phosphatidylinositol 3-kinase/akt cassette regulates purine nucleotide synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is highly conserved throughout evolution and regulates cell size and survival and cell cycle progression. It regulates the latter by stimulating procession through G(1) and the G(1)/S phase transition. Entry into S phase requires an abundant supply of purine nucleotides, but the effect of the PI3K/Akt pathway on purine synthesis has not been studied. We now show that the PI3K/Akt cassette regulates both de novo and salvage purine nucleotide synthesis in insulin-responsive mouse mesenchymal cells. We found that serum and insulin stimulated de novo purine synthesis in serum-starved cells largely through PI3K/Akt signaling, and pharmacologic and genetic inhibition of PI3K/Akt reduced de novo synthesis by 75% in logarithmically growing cells. PI3K/Akt regulated early steps of de novo synthesis by modulating phosphoribosylpyrophosphate production by the non-oxidative pentose phosphate pathway and late steps by modulating activity of the bifunctional enzyme aminoimidazole-carboxamide ribonucleotide transformylase IMP cyclohydrolase, an enzyme not previously known to be regulated. The effects of PI3K/Akt on purine nucleotide salvage were likely through regulating phosphoribosylpyrophosphate availability. These studies define a new mechanism whereby the PI3K/Akt cassette functions as a master regulator of cellular metabolism and a key player in oncogenesis.

Crystal structure and function of 5-formaminoimidazole-4-carboxamide ribonucleotide synthetase from Methanocaldococcus jannaschii.

Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.

Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside.

Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside-induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention.