[1,2,4]Triazolo[1,5-a]pyrimidine derivatives: Structure-activity relationship study leading to highly selective ENPP1 inhibitors.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.

Structural basis of the UDP-diacylglucosamine pyrophosphohydrolase LpxH inhibition by sulfonyl piperazine antibiotics.

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3-phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.

Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity.

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal ß-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 µM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

Cytosolic geraniol and citronellol biosynthesis require a Nudix hydrolase in rose-scented geranium (Pelargonium graveolens).

Geraniol, citronellol and their esters are high-value acyclic monoterpenes used in food technology, perfumery and cosmetics. A major source of these compounds is the essential oil of rose-scented geraniums of the genus Pelargonium. We provide evidence that their biosynthesis mainly takes place in the cytosol of glandular trichomes via geranyl monophosphate (GP) through the action of a Nudix hydrolase. Protein preparations could convert geranyl diphosphate (GDP) to geraniol in in vitro assays, a process which could be blocked by inorganic phosphatase inhibitors, suggesting a two-step conversion of GDP to geraniol. Pelargonium graveolens chemotypes enriched in either geraniol or (-)-citronellol accumulate GP or citronellyl monophosphate (CP), respectively, the presumed precursors to their monoterpenoid end products. Geranyl monophosphate was highly enriched in isolated glandular trichomes of lines producing high amounts of geraniol. In contrast, (-)-isomenthone-rich lines are depleted in these prenyl monophosphates and monoterpene alcohols and instead feature high levels of GDP, the precursor to plastidic p-menthane biosynthesis. A Nudix hydrolase cDNA from Pelargonium glandular trichomes, dubbed PgNdx1, encoded a cytosolic protein capable of hydrolyzing GDP to GP with a KM of about 750 nm but is only weakly active towards farnesyl diphosphate. In citronellol-rich lines, GDP, GP and CP were detected in nearly equimolar amounts, while citronellyl diphosphate was absent, suggesting that citronellol biosynthesis may proceed by reduction of GP to CP in this species. These findings highlight the cytosol as a compartment that supports monoterpene biosynthesis and expands the roles of Nudix hydrolases in the biosynthesis of plant volatiles.

Structure- and Ligand-Dynamics-Based Design of Novel Antibiotics Targeting Lipid A Enzymes LpxC and LpxH in Gram-Negative Bacteria.

Bacterial infections caused by multi-drug-resistant Gram-negative pathogens pose a serious threat to public health. Gram-negative bacteria are characterized by the enrichment of lipid A-anchored lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in the outer leaflet of their outer membrane. Constitutive biosynthesis of lipid A via the Raetz pathway is essential for bacterial viability and fitness in the human host. The inhibition of early-stage lipid A enzymes such as LpxC not only suppresses the growth of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter spp., and other clinically important Gram-negative pathogens but also sensitizes these bacteria to other antibiotics. The inhibition of late-stage lipid A enzymes such as LpxH is uniquely advantageous because it has an extra mechanism of bacterial killing through the accumulation of toxic lipid A intermediates, rendering LpxH inhibition additionally lethal to Acinetobacter baumannii. Because essential enzymes of the Raetz pathway have never been exploited by commercial antibiotics, they are excellent targets for the development of novel antibiotics against multi-drug-resistant Gram-negative infections.This Account describes the ongoing research on characterizing the structure and inhibition of LpxC and LpxH, the second and fourth enzymes of the Raetz pathway of lipid A biosynthesis, in the laboratories of Dr. Pei Zhou and Dr. Jiyong Hong at Duke University. Our studies have elucidated the molecular basis of LpxC inhibition by the first broad-spectrum inhibitor, CHIR-090, as well as the mechanism underlying its spectrum of activity. Such an analysis has provided a molecular explanation for the broad-spectrum antibiotic activity of diacetylene-based LpxC inhibitors. Through the structural and biochemical investigation of LpxC inhibition by diacetylene LpxC inhibitors and the first nanomolar LpxC inhibitor, L-161,240, we have elucidated the intrinsic conformational and dynamics difference in individual LpxC enzymes near the active site. A similar approach has been taken to investigate LpxH inhibition, leading to the establishment of the pharmacophore model of LpxH inhibitors and subsequent structural elucidation of LpxH in complex with its first reported small-molecule inhibitor based on a sulfonyl piperazine scaffold.Intriguingly, although our crystallographic analysis of LpxC- and LpxH-inhibitor complexes detected only a single inhibitor conformation in the crystal lattice, solution NMR studies revealed the existence of multiple ligand conformations that together delineate a cryptic ligand envelope expanding the ligand-binding footprint beyond that observed in the crystal structure. By harnessing the ligand dynamics information and structural insights, we demonstrate the feasibility to design potent LpxC and LpxH inhibitors by merging multiple ligand conformations. Such an approach has enabled us to rationally design compounds with significantly enhanced potency in enzymatic assays and outstanding antibiotic activities in vitro and in animal models of bacterial infection. We anticipate that continued efforts with structure and ligand dynamics-based lead optimization will ultimately lead to the discovery of LpxC- and LpxH-targeting clinical antibiotics against a broad range of Gram-negative pathogens.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Design, synthesis and biological evaluation studies of novel small molecule ENPP1 inhibitors for cancer immunotherapy.

Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents.

Oxidized DNA Precursors Cleanup by NUDT1 Contributes to Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.