Perspectives and challenges in developing small molecules targeting purine nucleoside phosphorylase.

As a cytosolic enzyme involved in the purine salvage pathway metabolism, purine nucleoside phosphorylase (PNP) plays an important role in a variety of cellular functions but also in immune system, including cell growth, apoptosis and cancer development and progression. Based on its T-cell targeting profile, PNP is a potential target for the treatment of some malignant T-cell proliferative cancers including lymphoma and leukemia, and some specific immunological diseases. Numerous small-molecule PNP inhibitors have been developed so far. However, only Peldesine, Forodesine and Ulodesine have entered clinical trials and exhibited some potential for the treatment of T-cell leukemia and gout. The most recent direction in PNP inhibitor development has been focused on PNP small-molecule inhibitors with better potency, selectivity, and pharmacokinetic property. In this perspective, considering the structure, biological functions, and disease relevance of PNP, we highlight the recent research progress in PNP small-molecule inhibitor development and discuss prospective strategies for designing additional PNP therapeutic agents.

[Methylthioadenosine phosphorylase and p16 as surrogate diagnostic markers for CDKN2A homozygous deletion in brain tumors].

Objective: To examine whether immunohistochemistry of methylthioadenosine phosphorylase (MTAP) and p16 could be used to predict the CDKN2A status in various brain tumors. Methods: A total of 118 cases of IDH-mutant astrocytomas, 16 IDH-wildtype glioblastoma, 17 polymorphic xanthoastrocytoma (PXA) and 20 meningiomas diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from November 2017 to October 2023 were collected and analyzed. The CDKN2A status was detected by using fluorescence in situ hybridization or next-generation sequencing. Expression of MTAP and p16 proteins was detected with immunohistochemistry. The association of loss of MTAP/p16 expression with CDKN2A homozygous/heterozygous deletion was examined. Results: Among the 118 cases of IDH-mutant astrocytoma, 13 cases showed homozygous deletion of CDKN2A. All of them had no expression of MTAP while 9 cases had no expression of p16. Among the 16 cases of IDH wild-type glioblastoma, 6 cases showed homozygous deletion of CDKN2A. All 6 cases had no expression of MTAP, while 3 of these cases had no expression of p16 expression. Among the 17 PXA cases, 4 cases showed homozygous deletion of CDKN2A, and the expression of MTAP and p16 was also absent in these 4 cases. Among the 20 cases of meningiomas, 4 cases showed homozygous deletion of CDKN2A. Their expression of MTAP and p16 was also absent. Among the four types of brain tumors, MTAP was significantly correlated with CDKN2A homozygous deletion (P<0.05), with a sensitivity of 100%. However, it was only significantly correlated with the loss of heterozygosity (LOH) of CDKN2A in astrocytomas (P<0.001). P16 was associated with CDKN2A homozygous deletion in IDH-mutant astrocytoma and PXA (P<0.001), but not with the LOH of CDKN2A. Its sensitivity and specificity were lower than that of MTAP. Conclusions: MTAP could serve as a predictive surrogate for CDKN2A homozygous deletion in adult IDH-mutant astrocytoma, PXA, adult IDH-wildtype glioblastoma and meningioma. However, p16 could only be used in the first two tumor types, and its specificity and sensitivity are lower than that of MTAP.

Loss of methylthioadenosine phosphorylase immunoreactivity correlates with poor prognosis and elevated uptake of 11C-methionine in IDH-mutant astrocytoma.

PURPOSE: The proximate localization of MTAP, which encodes methylthioadenosine phosphorylase, and CDKN2A/B on Chromosome 9q21 has allowed the loss of MTAP expression as a surrogate for homozygous deletion of CDKN2A/B. This study aimed to determine whether MTAP status correlates with clinical outcomes and 11C-methionine uptake in astrocytomas with IDH mutations. METHODS: We conducted immunohistochemistry for MTAP in 30 patients with astrocytoma, IDH-mutant who underwent 11C-methionine positron emission tomography scans prior to surgical resection. The tumor-to-normal (T/N) ratio of 11C-methionine uptake was calculated using the mean standardized uptake value (SUV) for tumor and normal brain tissues. Cox regression analysis was used for multivariate survival analysis. RESULTS: Among IDH-mutant astrocytomas, 26.7% (8/30) exhibited the loss of cytoplasmic MTAP expression, whereas 73.3% (22/30) tumors retained MTAP expression. The median progression-free survival (PFS) was significantly shorter in patients with MTAP loss than those with MTAP retention (1.88 years vs. 6.80 years, p = 0.003). The median overall survival (OS) was also shorter in patients with MTAP loss than in MTAP-retaining counterparts (5.23 years vs. 10.69 years, p = 0.019). Multivariate analysis identified MTAP status (hazard ratio (HR), 0.081) and extent of resection (HR, 0.104) as independent prognostic factors for PFS. Astrocytomas lacking cytoplasmic MTAP expression showed a significantly higher median T/N ratio for 11C-methionine uptake than tumors retaining MTAP (2.12 vs. 1.65, p = 0.012). CONCLUSION: Our study revealed that the loss of MTAP expression correlates with poor prognosis and an elevated T/N ratio of 11C-methionine uptake in astrocytoma, IDH-mutant.

Spermidine attenuates monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting purine metabolism and polyamine synthesis-associated vascular remodeling.

Ensuring the homeostatic integrity of pulmonary artery endothelial cells (PAECs) is essential for combatting pulmonary arterial hypertension (PAH), as it equips the cells to withstand microenvironmental challenges. Spermidine (SPD), a potent facilitator of autophagy, has been identified as a significant contributor to PAECs function and survival. Despite SPD's observed benefits, a comprehensive understanding of its protective mechanisms has remained elusive. Through an integrated approach combining metabolomics and molecular biology, this study uncovers the molecular pathways employed by SPD in mitigating PAH induced by monocrotaline (MCT) in a Sprague-Dawley rat model. The study demonstrates that SPD administration (5 mg/kg/day) significantly corrects right ventricular impairment and pathological changes in pulmonary tissues following MCT exposure (60 mg/kg). Metabolomic profiling identified a purine metabolism disorder in MCT-treated rats, which SPD effectively normalized, conferring a protective effect against PAH progression. Subsequent in vitro analysis showed that SPD (0.8 mM) reduces oxidative stress and apoptosis in PAECs challenged with Dehydromonocrotaline (MCTP, 50 µM), likely by downregulating purine nucleoside phosphorylase (PNP) and modulating polyamine biosynthesis through alterations in S-adenosylmethionine decarboxylase (AMD1) expression and the subsequent production of decarboxylated S-adenosylmethionine (dcSAM). These findings advocate SPD's dual inhibitory effect on PNP and AMD1 as a novel strategy to conserve cellular ATP and alleviate oxidative injuries, thus providing a foundation for SPD's potential therapeutic application in PAH treatment.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers.

BACKGROUND: One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. MATERIALS AND METHODS: We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. RESULTS: The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). CONCLUSION: In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

Polyamine Inhibitor SAM486A Augments Cytarabine Cytotoxicity in Methylthioadenosine Phosphorylase-deficient Leukemia Cells.

BACKGROUND/AIM: Methionine metabolism contributes to supplying sulfur-containing amino acids, controlling the methyl group transfer reaction, and producing polyamines in cancer cells. Polyamines play important roles in various cellular functions. Methylthioadenosine phosphorylase (MTAP), the key enzyme of the methionine salvage pathway, is reported to be deficient in 15-62% of cases of hematological malignancies. MTAP-deficient cancer cells accumulate polyamines, resulting in enhanced cell proliferation. The aim of this study was to investigate the combined effects of the polyamine synthesis inhibitor SAM486A and the anticancer antimetabolite cytarabine in MTAP-deficient leukemic cells in vitro. MATERIALS AND METHODS: The leukemia cell line U937 and the subline, U937/MTAP(-), in which MTAP was knocked down by shRNA, were used. The experiments were performed in media supplemented with 20% methionine (low methionine), which was the minimum concentration for maintaining cellular viability. RESULTS: The knockdown efficiency test confirmed a 70% suppression of the expression of the MTAP gene in U937/MTAP(-) cells. Even in the media with low methionine, the intracellular methionine concentration was not reduced in U937/MTAP(-) cells, suggesting that the minimum supply of methionine was sufficient to maintain intracellular levels of methionine. Both U937/MTAP(+) and U937/MTAP(-) cells were comparably sensitive to anticancer drugs (cytarabine, methotrexate, clofarabine and 6-thioguanine). The combination of SAM486A and cytarabine was demonstrated to have synergistic cytotoxicity in U937/MTAP(-) cells with regard to cell growth inhibition and apoptosis induction, but not in U937/MTAP(+) cells. Mechanistically, SAM486A altered the intracellular polyamine concentrations and reduced the antiapoptotic proteins. CONCLUSION: Methionine metabolism and polyamine synthesis can be attractive therapeutic targets in leukemia.

Purine nucleoside phosphorylase inhibition is an effective approach for the treatment of chemical hemorrhagic cystitis.

Hemorrhagic cystitis may be induced by infection, radiation therapy, or medications or may be idiopathic. Along with hemorrhagic features, symptoms include urinary urgency and frequency, dysuria (painful urination), and visceral pain. Cystitis-induced visceral pain is one of the most challenging types of pain to treat, and an effective treatment would address a major unmet medical need. We assessed the efficacy of a purine nucleoside phosphorylase inhibitor, 8-aminoguanine (8-AG), for the treatment of hemorrhagic/ulcerative cystitis. Lower urinary tract (LUT) function and structure were assessed in adult Sprague-Dawley rats, treated chronically with cyclophosphamide (CYP; sacrificed day 8) and randomized to daily oral treatment with 8-AG (begun 14 days prior to CYP induction) or its vehicle. CYP-treated rats exhibited multiple abnormalities, including increased urinary frequency and neural mechanosensitivity, reduced bladder levels of inosine, urothelial inflammation/damage, and activation of spinal cord microglia, which is associated with pain hypersensitivity. 8-AG treatment of CYP-treated rats normalized all observed histological, structural, biochemical, and physiological abnormalities. In cystitis 8-AG improved function and reduced both pain and inflammation likely by increasing inosine, a tissue-protective purine metabolite. These findings demonstrate that 8-AG has translational potential for reducing pain and preventing bladder damage in cystitis-associated LUT dysfunctions.

The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

BACKGROUND: Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. METHODS: In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. RESULTS: Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n = 13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n = 13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n = 4). CONCLUSIONS: Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Forodesine Enhances Immune Responses through Guanosine-Mediated TLR7 Activation while Preventing Graft-versus-Host Disease.

Recent evidence indicates that specific types of nuclear acids, including guanosine and its derivatives, act as natural ligands for TLR7. This led us to hypothesize that purine nucleoside phosphorylase inhibitors not only can induce apoptosis of T cells but also can lead to TLR7 activation by accumulation of guanine nucleosides, in particular under systemic inflammation, where damaged tissues release a large amount of nucleotides. We demonstrate in the present study that a purine nucleoside phosphorylase inhibitor, forodesine, can reduce the disease severity and prolong the survival in a xenogeneic mouse model of graft-versus-host disease (GVHD). Guanine nucleosides were undetectable in mice during GVHD but increased significantly following forodesine treatment. Our in vitro experiments showed that forodesine enhanced guanosine-mediated cytokine production from APCs, including alveolar macrophages and plasmacytoid dendritic cells, through TLR7 signaling. Forodesine also enhanced Ag-presenting capacity, as demonstrated by increased CD8+ T cell proliferation and higher secretion of IFN-γ and IL-12p40 in an MLR with plasmacytoid dendritic cells. Furthermore, forodesine stimulated IFN-γ production from activated T cells in the presence of a low concentration of guanosine while inhibiting their proliferation and inducing apoptotic cell death. Although forodesine ameliorated GVHD severity, mice treated with forodesine showed significantly higher levels of multiple proinflammatory cytokines and chemokines in plasma, suggesting in vivo upregulation of TLR7 signaling. Our study suggests that forodesine may activate a wide range of immune cells, including T cells, through TLR7 stimulation while inhibiting GVHD by inducing apoptosis of T cells, after allogeneic hematopoietic stem cell transplant.

Reliability assessment of methylthioadenosine phosphorylase immunohistochemistry as a surrogate biomarker for CDKN2A homozygous deletion in adult-type IDH-mutant diffuse gliomas.

According to the 2021 World Health Organization classification of brain tumors, astrocytomas containing a CDKN2A/B homozygous deletion (HD) are designated as grade 4 even when no microvascular proliferation and/or necrosis is present. In this study, we aimed to investigate the relationship between CDKN2A HD and loss of methylthioadenosine phosphorylase (MTAP) expression in adult-type IDH-mutant gliomas and to assess the sensitivity and specificity of MTAP immunohistochemistry (IHC) along with interobserver agreement as a surrogate biomarker for CDKN2A HD. Eighty-eight astrocytomas and 71 oligodendrogliomas cases that were diagnosed between 2014 and 2021 at Hacettepe University were selected and tissue microarrays were conducted to perform CDKN2A fluorescence in situ hybridization and MTAP IHC. Twenty-five (15.7%) cases harbored CDKN2A HD. MTAP loss was detected in 28 (15.7%) cases by the first observer and 27 (17%) cases by the second observer. The sensitivity and specificity of MTAP were calculated as 88% and 95.52%-96.27% for 2 observers. A very good/perfect agreement was noted between the observers (Cohen kappa coefficient = 0.938). Intratumoral heterogeneity was observed in 4 cases. MTAP IHC was found to be a reliable surrogate biomarker as a possible alternative to CDKN2A HD identification with a high sensitivity and specificity along with high interobserver agreement.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites. A global effort to identify lead compounds led to several initiatives such as the Medicine for Malaria Ventures (MMV), a repository of compounds showing promising efficacy in killing the parasite in cell-based assays. Here, we used mass spectrometry coupled with cellular thermal shift assay to identify putative protein targets of MMV000848, a compound with an in vitro EC50 of 0.5 µM against the parasite. Thermal shift assays showed a strong increase of P. falciparum purine nucleoside phosphorylase (PfPNP) melting temperature by up to 15 °C upon incubation with MMV000848. Binding and enzymatic assays returned a KD of 1.52 ± 0.495 µM and an IC50 value of 21.5 ± 2.36 µM. The inhibition is competitive with respect to the substrate, as confirmed by a cocrystal structure of PfPNP bound with MMV000848 at the active site, determined at 1.85 Å resolution. In contrast to transition states inhibitors, MMV000848 specifically inhibits the parasite enzyme but not the human ortholog. An isobologram analysis shows subadditivity with immucillin H and with quinine respectively, suggesting overlapping modes of action between these compounds. These results point to PfPNP as a promising antimalarial target and suggest avenues to improve inhibitor potency.

SERS analysis of cancer cell-secreted purines reveals a unique paracrine crosstalk in MTAP-deficient tumors.

The tumor microenvironment (TME) is a dynamic pseudoorgan that shapes the development and progression of cancers. It is a complex ecosystem shaped by interactions between tumor and stromal cells. Although the traditional focus has been on the paracrine communication mediated by protein messengers, recent attention has turned to the metabolic secretome in tumors. Metabolic enzymes, together with exchanged substrates and products, have emerged as potential biomarkers and therapeutic targets. However, traditional techniques for profiling secreted metabolites in complex cellular contexts are limited. Surface-enhanced Raman scattering (SERS) has emerged as a promising alternative due to its nontargeted nature and simplicity of operation. Although SERS has demonstrated its potential for detecting metabolites in biological settings, its application in deciphering metabolic interactions within multicellular systems like the TME remains underexplored. In this study, we introduce a SERS-based strategy to investigate the secreted purine metabolites of tumor cells lacking methylthioadenosine phosphorylase (MTAP), a common genetic event associated with poor prognosis in various cancers. Our SERS analysis reveals that MTAP-deficient cancer cells selectively produce methylthioadenosine (MTA), which is taken up and metabolized by fibroblasts. Fibroblasts exposed to MTA exhibit: i) molecular reprogramming compatible with cancer aggressiveness, ii) a significant production of purine derivatives that could be readily recycled by cancer cells, and iii) the capacity to secrete purine derivatives that induce macrophage polarization. Our study supports the potential of SERS for cancer metabolism research and reveals an unprecedented paracrine crosstalk that explains TME reprogramming in MTAP-deleted cancers.

Biodegradation of Inosine and Guanosine by Bacillus paranthracis YD01.

Both inosine and guanosine are precursors of uric acid that may cause the diseases of hyperuricemia and gout in humans. Here, a promising bacterial strain for efficiently biodegrading both inosine and guanosine was successfully isolated from a healthy human intestine and identified as Bacillus paranthracis YD01 with 16S rRNA analysis. An initial amount of 49.6 mg·L-1 of inosine or 49.9 mg·L-1 of guanosine was completely removed by YD01 within 12 h, which showed that YD01 had a strong ability to biodegrade inosine and guanosine. Furthermore, the initial amount of 49.2 mg·L-1 of inosine or 49.5 mg·L-1 of guanosine was totally catalyzed by the intracellular crude enzymes of YD01 within 6 h, and the initial inosine amount of 49.6 mg·L-1 or guanosine of 49.7 mg·L-1 was biodegraded by the extracellular crude enzymes of YD01 within 9 h. Illumina Hiseq sequencing and database gene annotation were used to elucidate the genomic characteristics of B. paranthracis YD01. Purine nucleoside phosphorylase, encoded by gene 1785, gene 3933, and gene 4403, was found in the KEEG database, which played a crucial role in the biodegradation of inosine and guanosine. The results of this study provide valuable insights into the mechanisms for biodegrading inosine and guanosine using B. paranthracis YD01.

Decreased Transition-State Analogue Affinity in Isotopically Heavy MTAN with Increased Catalysis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase from Helicobacter pylori (HpMTAN) demonstrated faster chemistry when expressed as an isotopically heavy protein, with 2H, 13C, and 15N replacing the bulk of normal isotopes. The inverse heavy enzyme isotope effect has been attributed to improved enzyme-reactant interactions causing more frequent transition-state formation ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2109118118). Transition-state analogues stabilize the transient dynamic geometry of the transition state and inform on transition-state dynamics. Here, a slow-onset, tight-binding transition-state analogue of HpMTAN is characterized with heavy and light enzymes. Dissociation constants for the initial encounter complex (Ki) and for the tightly bound complex after slow-onset inhibition (Ki*) with hexylthio-DADMe-Immucillin-A (HTDIA) gave Ki values for light and heavy HpMTAN = 52 ± 10 and 85 ± 13 pM and Ki* values = 5.9 ± 0.3 and 10.0 ± 1.2 pM, respectively. HTDIA dissociates from heavy HpMTAN at 0.063 ± 0.002 min-1, faster than that from light HpMTAN at 0.032 ± 0.004 min-1. These values are consistent with transition-state formation by an improved catalytic site dynamic search and inconsistent with catalytic efficiency proportional to tight binding of the transition state.

Phosphate Binding in PNP Alters Transition-State Analogue Affinity and Subunit Cooperativity.

Purine nucleoside phosphorylases (PNPs) catalyze the phosphorolysis of 6-oxypurine nucleosides with an HPO42- dianion nucleophile. Nucleosides and phosphate occupy distinct pockets in the PNP active site. Evaluation of the HPO42- site by mutagenesis, cooperative binding studies, and thermodynamic and structural analysis demonstrate that alterations in the HPO42- binding site can render PNP inactive and significantly impact subunit cooperativity and binding to transition-state analogue inhibitors. Cooperative interactions between the cationic transition-state analogue and the anionic HPO42- nucleophile demonstrate the importance of reforming the transition-state ensemble for optimal inhibition with transition-state analogues. Altered phosphate binding in the catalytic site mutants helps to explain one of the known lethal PNP deficiency syndromes in humans.

Enzyme-mediated depletion of methylthioadenosine restores T cell function in MTAP-deficient tumors and reverses immunotherapy resistance.

Chromosomal region 9p21 containing tumor suppressors CDKN2A/B and methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic deletions in cancer. 9p21 loss is correlated with reduced tumor-infiltrating lymphocytes (TILs) and resistance to immune checkpoint inhibitor (ICI) therapy. Previously thought to be caused by CDKN2A/B loss, we now show that it is loss of MTAP that leads to poor outcomes on ICI therapy and reduced TIL density. MTAP loss causes accumulation of methylthioadenosine (MTA) both intracellularly and extracellularly and profoundly impairs T cell function via the inhibition of protein arginine methyltransferase 5 (PRMT5) and by adenosine receptor agonism. Administration of MTA-depleting enzymes reverses this immunosuppressive effect, increasing TILs and drastically impairing tumor growth and importantly, synergizes well with ICI therapy. As several studies have shown ICI resistance in 9p21/MTAP null/low patients, we propose that MTA degrading therapeutics may have substantial therapeutic benefit in these patients by enhancing ICI effectiveness.

Neurologic Status of Patients with Purine Nucleoside Phosphorylase Deficiency Before and After Hematopoetic Stem Cell Transplantation.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive combined immunodeficiency. The phenotype is profound T cell deficiency with variable B and NK cell functions and results in recurrent and persistent infections that typically begin in the first year of life. Neurologic findings occur in approximately two-thirds of patients. The mechanism of neurologic abnormalities is unclear. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for PNP deficiency. METHODS: We report here six patients from five unrelated families with PNP deficiency treated in two centers in Turkey. We evaluated the neurological status of patients and compared to post-transplantation period if available. Then, we performed PubMed, Google Scholar, and Researchgate searches using the terms "PNP" and "hematopoietic stem cell transplantation" to find all reported cases of PNP transplantation and compared to our cohort. RESULTS: Six patients were treated in two centers in Turkey. One patient died from post-transplant complications. The other four patients underwent successful HSCT with good immune reconstitution after transplantation (follow-up 21-48 months) and good neurological outcomes. The other patient with a new mutation is still waiting for a matching HLA donor. DISCUSSION: In PNP deficiency, clinical manifestations are variable, and this disease should be considered in the presence of many different clinical findings. Despite the comorbidities that occurred before transplantation, HSCT currently appears to be the only treatment option for this disease. HSCT not only cures immunologic disorders, but probably also improves or at least stabilizes the neurologic status of patients.

Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum.

2'-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2'-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2'-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2'-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.

Investigating the Vital Role of the Identified Abietic Acid from Helianthus annuus L. Calathide Extract against Hyperuricemia via Human Embryonic Kidney 293T Cell Model.

To explore the anti-hyperuricemia components in sunflower (Helianthus annuus L.) calathide extract (SCE), we identified abietic acid (AA) via liquid chromatography-mass spectrometry and found an excellent inhibitor of xanthine oxidase (IC50 = 10.60 µM, Ki = 193.65 nM) without cytotoxicity. Based on the transcriptomics analysis of the human embryonic kidney 293T cell model established using 1 mM uric acid, we evaluated that AA showed opposite modulation of purine metabolism to the UA group and markedly suppressed the intensity of purine nucleoside phosphorylase, ribose phosphate pyrophosphokinase 2, and ribose 5-phosphate isomerase A. Molecular docking also reveals the inhibition of purine nucleoside phosphorylase and ribose phosphate pyrophosphokinase 1. The SCE exhibits similar regulation of these genes, so we conclude that AA was a promising component in SCE against hyperuricemia. This present study provided a novel cell model for screening anti-hyperuricemia natural drugs in vitro and illustrated that AA, a natural diterpenoid, is a potential inhibitor of purine biosynthesis or metabolism.