Discovery of a spirocyclic 3-bromo-4,5-dihydroisoxazole covalent inhibitor of hGAPDH with antiproliferative activity against pancreatic cancer cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Covalent inhibitors of GAPDH: From unspecific warheads to selective compounds.

Targeting glycolysis is an attractive approach for the treatment of a wide range of pathologies, such as various tumors and parasitic infections. Due to its pivotal role in the glycolysis, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this pathway for energy production. In this context, GAPDH inhibition can be a valuable approach for the development of anticancer and antiparasitic drugs. In addition to its glycolytic role, GAPDH possesses several moonlight functions, whose deregulation is involved in some pathological conditions. Covalent modification on different amino acids of GAPDH, in particular on cysteine residues, can lead to a modulation of the enzyme activity. The selectivity towards specific cysteine residues is essential to achieve a specific phenotypic effect. In this work we report an extensive overview of the latest advances on the numerous compounds able to inhibit GAPDH through the covalent binding to cysteine residues, ranging from endogenous metabolites and xenobiotics, which may serve as pharmacological tools to actual drug-like compounds with promising therapeutic perspectives. Furthermore, we focused on the potentialities of the different warheads, shedding light on the possibility to exploit a combination of a finely tuned electrophilic group with a well-designed recognition moiety. These findings can provide useful information for the rational design of novel covalent inhibitors of GAPDH, with the final goal to expand the current treatment options.

Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign.

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives.

Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. METHODS: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. RESULTS: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. CONCLUSION: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.

Mutant p53 prevents GAPDH nuclear translocation in pancreatic cancer cells favoring glycolysis and 2-deoxyglucose sensitivity.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies. In about 70% of PDACs the tumor suppressor gene TP53 is mutated generally resulting in conformational changes of mutant p53 (mutp53) proteins, which acquire oncogenic functions triggering aggressiveness of cancers and alteration of energetic metabolism. Here, we demonstrate that mutant p53 prevents the nuclear translocation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) stabilizing its cytoplasmic localization, thus supporting glycolysis of cancer cells and inhibiting cell death mechanisms mediated by nuclear GAPDH. We further show that the prevention of nuclear localization of GAPDH is mediated by both stimulation of AKT and repression of AMPK signaling, and is associated with the formation of the SIRT1:GAPDH complex. By using siRNA-GAPDH or an inhibitor of the enzyme, we functionally demonstrate that the maintenance of GAPDH in the cytosol has a critical impact on the anti-apoptotic and anti-autophagic effects driven by mutp53. Furthermore, the blockage of its mutp53-dependent cytoplasmic stabilization is able to restore the sensitivity of PDAC cells to the treatment with gemcitabine. Finally, our data suggest that mutp53-dependent enhanced glycolysis permits cancer cells to acquire sensitivity to anti-glycolytic drugs, such as 2-deoxyglucose, suggesting a potential personalized therapeutic approach in human cancers carrying mutant TP53 gene.

UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism.

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenases.

Compounds based on the 3-Br-isoxazoline scaffold fully inhibit glyceraldehyde 3-phosphate dehydrogenase from Plasmodium falciparum by selectively alkylating all four catalytic cysteines of the tetramer. Here, we show that, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human ortholog was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer. The partial alkylation seems to produce a slow conformational rearrangement that severely limits the accessibility of the remaining active sites to bulky 3-Br-isoxazoline derivatives, but not to the substrate or smaller alkylating agents.

Inspired by Nature: The 3-Halo-4,5-dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors.

Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re-emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3-chloro-4,5-dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3-halo-4,5-dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3-halo-4,5-dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.

Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity.

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 µM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 µM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.

Synthesis and biological evaluation of papain-family cathepsin†L-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents.

Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3-bromoisoxazoline moiety. Several rhodesain and falcipain-2 inhibitors displaying single-digit micromolar or sub-micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.

Development of rhodesain inhibitors with a 3-bromoisoxazoline warhead.

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3-bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar Ki values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC50 values in the mid-micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.

Inhibition of rhodesain as a novel therapeutic modality for human African trypanosomiasis.

Rhodesain, a cathepsin L-like cysteine protease of T. brucei rhodesiense, is considered a potential target for the treatment of human African trypanosomiasis. Recent findings have confirmed that rhodesain, a lysosomal protease, is essential for parasite survival. Rhodesain is required by T. brucei to cross the blood-brain barrier, degrade host immunoglobulins, and turn over variant surface coat glycoproteins of T. brucei, which impair effective host immune responses. In this Perspective, we discuss the main classes of rhodesain inhibitors, including peptidic, peptidomimetic, and nonpeptidic structures, emphasizing those that have exhibited an optimal match between enzymatic affinity and trypanocidal profile and those for which preclinical investigations are currently in progress.

Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds.

Tumor resistance to antitubulin drugs resulting from P-glycoprotein (Pgp) drug-efflux activity, increased expression of the ßIII tubulin isotype, and alterations in the drug-binding sites are major obstacles in cancer therapy. Consequently, novel antitubulin drugs that overcome these challenges are of substantial interest. Here, we study a novel chemotype named furan metotica that localizes to the colchicine-binding site in ß-tubulin, inhibits tubulin polymerization, and is not antagonized by Pgp. To elucidate the structure-activity properties of this chiral chemotype, the enantiomers of its most potent member were separated and their absolute configurations determined by X-ray crystallography. Both isomers were active and inhibited all 60 primary cancer cell lines tested at the U.S. National Cancer Institute. They also efficiently killed drug-resistant cancer cells that overexpressed the Pgp drug-efflux pump 10(6)-fold. In vitro, the R-isomer inhibited tubulin polymerization at least 4-fold more potently than the S-isomer, whereas in human cells the difference was 30-fold. Molecular modeling showed that the two isomers bind to ß-tubulin in distinct manners: the R-isomer binds in a colchicine-like mode and the S-isomer in a podophyllotoxin-like fashion. In addition, the dynamic binding trajectory and occupancy state of the R-isomer were energetically more favorable then those of the S-isomer, explaining the observed differences in biologic activities. The ability of a racemic drug to assume the binding modes of two prototypical colchicine-site binders represents a novel mechanistic basis for antitubulin activity and paves the way toward a comprehensive design of novel anticancer agents.

Synthesis and biochemical evaluation of δ(2)-isoxazoline derivatives as DNA methyltransferase 1 inhibitors.

A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 µM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.

Design and synthesis of novel isoxazole-based HDAC inhibitors.

A series of isoxazole-based histone deacetylase (HDAC) inhibitors structurally related to SAHA were designed and synthesized. The isoxazole moiety was inserted in the vicinity of the Zn(2+)-binding group in order to check its participation in the coordinating process.

Synthesis and biological evaluation of new amino acids structurally related to the antitumor agent acivicin.

A set of racemic conformationally constrained analogues of the antitumor antibiotic acivicin (+)-1 has been prepared through a strategy based on 1,3-dipolar cycloaddition of bromonitrile oxide to suitable dipolarophiles. The bromo analogue (2) of acivicin was also synthesized and tested as a reference compound, together with its stereoisomer 3. The antitumor properties of novel amino acids 4-7 were evaluated in vitro against human tumor cell lines. Their efficacy to inhibit glutamate synthase (GltS) from Azospirillum brasilense was also assayed. None of the studied compounds, but 2, showed significant activity.