Development of novel synthesized phthalazinone-based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer.

Herein we report the synthesis of two series of 4-phenylphthalazin-1-ones 11a-i and 4- benzylphthalazin-1-ones 16a-h as anti-lung adenocarcinoma agents with potential inhibitory activity against PARP-1. All the newly synthesized phthalazinones were evaluated for their anti-proliferative activity against A549 lung carcinoma cell line. Phthalazinones 11c-i and 16b, c showed significant cytotoxic activity against A549 cells at different concentrations (0.1, 1 and 10 µM) for two time intervals (24 h and 48 h). These nine phthalazinones were further examined for their inhibitory activity towards PARP-1. Compound 11c emerged as the most potent PARP-1 inhibitor with IC50 value of 97 nM, compared to that of Olaparib (IC50 = 139 nM). Furthermore, all these nine phthalazinones passed the filters of Lipinski and Veber rules, and predicted to have good pharmacokinetics properties in a theoretical kinetic study. On the other hand, western blotting in A549 cells revealed the enhanced expression of the cleaved PARP-1, alongside, with the reduced expression of pro-caspase-3 and phosphorylated AKT. In addition, ELISA assay confirmed the up-regulation of active caspase-3 and caspase-9 levels compared to the control, suggesting the activation of the apoptotic machinery in the A549 cells. Finally, molecular docking of 11c into PARP-1 active site (PDB: 5WRZ) was performed to explore the probable binding mode.

Synthesis and evaluation of malonate-based inhibitors of phosphosugar-metabolizing enzymes: class II fructose-1,6-bis-phosphate aldolases, type I phosphomannose isomerase, and phosphoglucose isomerase.

In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.

Glycolytic and non-glycolytic functions of Mycobacterium tuberculosis fructose-1,6-bisphosphate aldolase, an essential enzyme produced by replicating and non-replicating bacilli.

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

Rational design, synthesis, and evaluation of new selective inhibitors of microbial class II (zinc dependent) fructose bis-phosphate aldolases.

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows K(i) against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 10(5)). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.

Highly Selective Inhibitors of Class II Microbial Fructose Bis-phosphate Aldolases.

We hereby describe the rationale synthesis and biochemical evaluation of the most powerful and selective inhibitors of class II fructose bis-phosphate aldolases so far reported. These inhibitors are of potential therapeutic interest, since the class II enzyme is present exclusively in microorganisms (among which many pathogenic species) and is absent from man, plants, and animals.

Synthesis and biochemical evaluation of selective inhibitors of class II fructose bisphosphate aldolases: towards new synthetic antibiotics.

We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating group. It is a powerful and highly selective competitive inhibitor of isolated class II aldolases. We report crystallographic studies of this inhibitor bound in the active site of the Helicobacter pylori enzyme. The compound also shows activity against Mycobacterium tuberculosis isolates.