Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands.

Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.

Molluscicidal and Cercaricidal Effects of Myrciaria floribunda Essential Oil Nanoemulsion.

Schistosomiasis is a tropical disease transmitted in an aqueous environment by cercariae from the Schistosoma genus. This disease affects 200 million people living in risk areas around the world. The control of schistosomiasis is realized by chemotherapy, wastewater sanitation, health education, and mollusk control using molluscicidal agents. This work evaluates the effects of a nanoemulsion containing essential oil from Myrciaria floribunda leaves as a molluscicidal and cercaricidal agent against Biomphalaria glabrata mollusks and Schistosoma mansoni cercariae. The Myrciaria floribunda essential oil from leaves showed nerolidol, ß-selinene, 1,8 cineol, and zonarene as major constituents. The formulation study suggested the F3 formulation as the most promising nanoemulsion with polysorbate 20 and sorbitan monooleate 80 (4:1) with 5% (w/w) essential oil as it showed a smaller droplet size of approximately 100 nm with a PDI lower than 0.3 and prominent bluish reflection. Furthermore, this nanoemulsion showed stability after 200 days under refrigeration. The Myrciaria floribunda nanoemulsion showed LC50 values of 48.11 µg/mL, 29.66 µg/mL, and 47.02 µg/mL in Biomphalaria glabrata embryos, juveniles, and adult mollusks, respectively, after 48 h and 83.88 µg/mL for Schistosoma mansoni cercariae after 2 h. In addition, a survival of 80% was observed in Danio rerio, and the in silico toxicity assay showed lower overall human toxicity potential to the major compounds in the essential oil compared to the reference molluscicide niclosamide. These results suggest that the nanoemulsion of Myrciaria floribunda leaves may be a promising alternative for schistosomiasis control.

In silico and pharmacological study of N,S-acetal juglone derivatives as inhibitors of the P2X7 receptor-promoted in vitro and in vivo inflammatory response.

Purinergic receptors are transmembrane proteins responsive to extracellular nucleotides and are expressed by several cell types throughout the human body. Among all identified subtypes, the P2×7 receptor has emerged as a relevant target for the treatment of inflammatory disease. Several clinical trials have been conducted to evaluate the effectiveness of P2×7R antagonists. However, to date, no selective antagonist has reached clinical use. In this work, we report the pharmacological evaluation of eleven N, S-acetal juglone derivatives as P2×7R inhibitors. Using in vitro assays and in vivo experimental models, we identified one derivative with promising inhibitory activity and low toxicity. Our in silico studies indicate that the 1,4-naphthoquinone moiety might be a valuable molecular scaffold for the development of novel P2×7R antagonists, as suggested by our previous studies.

Synthesis, Biological Evaluation and Molecular Modeling Studies of Naphthoquinone Sulfonamides and Sulfonate Ester Derivatives as P2X7 Inhibitors.

ATP acts in the extracellular environment as an important signal, activating a family of receptors called purinergic receptors. In recent years, interest in the potential therapeutics of purinergic components, including agonists and antagonists of receptors, has increased. Currently, many observations have indicated that ATP acts as an important mediator of inflammatory responses and, when found in high concentrations in the extracellular space, is related to the activation of the P2X7 purinergic receptor. In this sense, the search for new inhibitors for this receptor has attracted a great deal of attention in recent years. Sulfonamide derivatives have been reported to be potent inhibitors of P2X receptors. In this study, ten naphthoquinone sulfonamide derivatives and five naphthoquinone sulfonate ester derivatives were tested for their inhibitory activity on the P2X7 receptor expressed in peritoneal macrophages. Some compounds showed promising results, displaying IC50 values lower than that of A740003. Molecular docking and dynamic studies also indicated that the active compounds bind to an allosteric site on P2X7R. The binding free energy indicates that sulfonamides have an affinity for the P2X7 receptor similar to A740003. Therefore, the compounds studied herein present potential P2X7R inhibition.

Triazoles with inhibitory action on P2X7R impaired the acute inflammatory response in vivo and modulated the hemostatic balance in vitro and ex vivo.

OBJECTIVE: The present study aimed to investigate five triazole compounds as P2X7R inhibitors and evaluate their ability to reduce acute inflammation in vivo. MATERIAL: The synthetic compounds were labeled 5e, 8h, 9i, 11, and 12. TREATMENT: We administered 500 ng/kg triazole analogs in vivo, (1-10 µM) in vitro, and 1000 mg/kg for toxicological assays. METHODS: For this, we used in vitro experiments, such as platelet aggregation, in vivo experiments of paw edema and peritonitis in mice, and in silico experiments. RESULTS: The tested substances 5e, 8h, 9i, 11, and 12 produced a significant reduction in paw edema. Molecules 5e, 8h, 9i, 11, and 12 inhibited carrageenan-induced peritonitis. Substances 5e, 8h, 9i, 11, and 12 showed an anticoagulant effect, and 5e at a concentration of 10 µM acted as a procoagulant. All derivatives, except for 11, had pharmacokinetic, physicochemical, and toxicological properties suitable for substances that are candidates for new drugs. In addition, the ADMET risk assessment shows that derivatives 8h, 11, 5e, and 9i have high pharmacological potential. Finally, docking tests indicated that the derivatives have binding energies comparable to the reference antagonist with a competitive inhibition profile. CONCLUSIONS: Together, the results indicate that the molecules tested as antagonist drugs of P2X7R had anti-inflammatory action against the acute inflammatory response.

Synthesis of new N,S-acetal analogs derived from juglone with cytotoxic activity against Trypanossoma cruzi.

A series of 11 new N,S-acetal juglone derivatives were synthesized and evaluated against T. cruzi epimastigote forms. These compounds were obtained in good to moderate yields using a microwave irradiation protocol. Among all compounds, two N,S-acetal analogs, showed significant trypanocidal activity. Notably, one compound 11g exhibited selectivity index 10-fold higher than the reference drug benznidazole for epimastigote. The compound 11h was more effective for amastigote forms. Both prototypes exhibited S.I. higher than the benznidazole description. Thus, both compounds proving to be useful candidate molecules to further studies in infected animals.

Arylboronic acids inhibit P2X7 receptor function and the acute inflammatory response.

The P2X7 receptor (P2X7R) is an ion channel which is activated by interactions with the extracellular ATP molecules. The molecular complex P2X7R/ATP induces conformational changes in the protein subunits, opening a pore in the ion channel macromolecular structure. Currently, the P2X7R has been studied as a potential therapeutic target of anti-inflammatory drugs. Based on this, a series of eight boronic acids (NO) analogs were evaluated on the biologic effect of this pharmacophoric group on the human and murine P2X7R. The boronic acids derivatives NO-01 and NO-12 inhibited in vitro human and murine P2X7R function. These analogs compounds showed effect better than compound BBG and similar to inhibitor A740003 for inhibiting dye uptake, in vitro IL-1ß release and ATP-induced paw edema in vivo. In both, in vitro and in vivo assays the compound NO-01 showed to be the hit compound in the present series of the arylboronic acids analogs. The molecular docking suggests that the NO derivatives bind into the upper body domain of the P2X7 pore and that the main intermolecular interaction with the two most active NO derivatives occur with the residues Phe 95, 103 and 293 by hydrophobic interactions and with Leu97, Gln98 and Ser101 by hydrogen bonds.. These results indicate that the boronic acid derivative NO-01 shows the lead compound characteristics to be used as a scaffold structure to the development of new P2X7R inhibitors with anti-inflammatory action.

1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole derivatives blockade P2X7 receptor in vitro and inflammatory response in vivo.

Fifty-one 1,2,3-triazole derivatives were synthesized and evaluated with respect to P2X7 receptor (P2X7R) activity and its associated pore. These triazoles were screened in vitro for dye uptake assay and its cytotoxicity against mammalian cell types. Seven 1,2,3-triazole derivatives (5e, 6e, 8h, 9d, 9i, 11, and 12) potently blocked P2X7 receptor pore formation in vitro (J774.G8 cells and peritoneal macrophages). All blockers displayed IC50 value inferior to 500 nM, and they have low toxicity in either cell types. These seven selected triazoles inhibited P2X7R mediated interleukin-1 (IL-1ß) release. In particular, compound 9d was the most potent P2X7R blocker. Additionally, in mouse acute models of inflammatory responses induced by ATP or carrageenan administration in the paw, compound 9d promoted a potent blocking response. Similarly, 9d also reduced mouse LPS-induced pleurisy cellularity. In silico predictions indicate this molecule appropriate to develop an anti-inflammatory agent when it was compared to commercial analogs. Electrophysiological studies suggest a competitive mechanism of action of 9d to block P2X7 receptor. Molecular docking was performed on the ATP binding site in order to observe the preferential interaction pose, indicating that binding mode of the 9d is by interacting its 1,2,3-triazole and ether moiety with positively charged residues and with its chlorobenzene moiety orientated toward the apolar end of the ATP binding site which are mainly composed by the Ile170, Trp167 and Leu309 residues from α subunit. These results highlight 9d derivative as a drug candidate with potential therapeutic application based on P2X7 receptor blockade.