Valence-driven colorimetric detection of norovirus protease via peptide-AuNP interactions.

We report here a colorimetric method for rapid detection of norovirus based on the valence-driven peptide-AuNP interactions. We engineered a peptide sequence named K1 with a cleavage sequence in between two lysine residues. The positively charged lysine groups aggregated the negatively charged nanoparticles leading to a purple color change. There was a red color when the cleavage sequence was digested by the Southampton norovirus 3C-like protease (SV3CP)-a protease involved in the life cycle of Human norovirus (HNV). The limit of detection was determined to be 320 nM in Tris buffer. We further show that the sensor has good performance in exhaled breath condensate, urine, and faecal matter. This research provides a potential easy and quick way to selectively detect HNV.

Structure-based discovery of thiosemicarbazones as SARS-CoV-2 main protease inhibitors.

Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 µM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.

Computer-aided design of 1,4-naphthoquinone-based inhibitors targeting cruzain and rhodesain cysteine proteases.

Chagas disease and Human African Trypanosomiasis (HAT) are caused by Trypanosoma cruzi and T. brucei parasites, respectively. Cruzain (CRZ) and Rhodesain (RhD) are cysteine proteases that share 70% of identity and play vital functions in these parasites. These macromolecules represent promising targets for designing new inhibitors. In this context, 26 CRZ and 5 RhD 3D-structures were evaluated by molecular redocking to identify the most accurate one to be utilized as a target. Posteriorly, a virtual screening of a library containing 120 small natural and nature-based compounds was performed on both of them. In total, 14 naphthoquinone-based analogs were identified, synthesized, and biologically evaluated. In total, five compounds were active against RhD, being three of them also active on CRZ. A derivative of 1,4-naphthoquinonepyridin-2-ylsulfonamide was found to be the most active molecule, exhibiting IC50 values of 6.3 and 1.8 µM for CRZ and RhD, respectively. Dynamic simulations at 100 ns demonstrated good stability and do not alter the targets' structures. MM-PBSA calculations revealed that it presents a higher affinity for RhD (-25.3 Kcal mol-1) than CRZ, in which van der Waals interactions were more relevant. A mechanistic hypothesis (via C3-Michael-addition reaction) involving a covalent mode of inhibition for this compound towards RhD was investigated by covalent molecular docking and DFT B3LYP/6-31 + G* calculations, exhibiting a low activation energy (ΔG‡) and providing a stable product (ΔG), with values of 7.78 and - 39.72 Kcal mol-1, respectively; similar to data found in the literature. Nevertheless, a reversibility assay by dilution revealed that JN-11 is a time-dependent and reversible inhibitor. Finally, this study applies modern computer-aided techniques to identify promising inhibitors from a well-known chemical class of natural products. Then, this work could inspire other future studies in the field, being useful for designing potent naphthoquinones as RhD inhibitors.

Desing and synthesis of potent anti-Trypanosoma cruzi agents new thiazoles derivatives which induce apoptotic parasite death.

Chagas disease, caused by the kinetoplastid protozoan parasite Trypanosoma cruzi, remains a relevant cause of illness and premature death and it is estimated that 6 million to 7 million people are infected worldwide. Although chemotherapy options are limited presenting serious problems, such as low efficacy and high toxicity. T. cruzi is susceptible to thiazoles, making this class of compounds appealing for drug development. Previously, thiazoles resulted in an increase in anti-T. cruzi activity in comparison to thiosemicarbazones. Here, we report the structural planning, synthesis and anti-T. cruzi evaluation of new thiazoles derivatives (3a-m and 4a-m), designed from molecular hybridization associated with non-classical bioisosterism. By varying substituents attached to the phenyl and thiazole rings, substituents were observed to retain, enhance or greatly increase their anti-T. cruzi activity, in comparison to the corresponding thiosemicarbazones. In most cases, electron-withdrawing substituents, such as bromine, 3,4-dichloro and nitro groups, greatly increased antiparasitic activity. Specifically, new thiazoles were identified that inhibit the epimastigote proliferation and were toxic for trypomastigotes without affecting macrophages viability. These compounds were also evaluated against cruzain. However, inhibition of this enzyme was not observed, suggesting that the compounds work through another mechanism. In addition, examination of T. cruzi cell death showed that these molecules induce apoptosis. In conclusion, except for compounds 3h and 3k, all thiazoles derivatives evaluated exhibited higher cytotoxic activity against the trypomastigote forms than the reference medicament benznidazole, without affecting macrophages viability. Compounds 4d and 4k were highlights, CC50 = 1.2 e 1.6 µM, respectively. Mechanistically, these compounds do not inhibit the cruzain, but induce T. cruzi cell death by an apoptotic process, being considered a good starting point for the development of new anti-Chagas drug candidates.

Synthesis and biological evaluation of potential inhibitors of the cysteine proteases cruzain and rhodesain designed by molecular simplification.

Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.

2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: structural design, synthesis and pharmacological evaluation.

The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a novel series of 2-(pyridin-2-yl)-1,3-thiazoles derived from 2-pyridine thiosemicarbazone. The majority of these compounds are potent cruzain inhibitors and showed excellent inhibition on the trypomastigote form of the parasite, and the resulting structure-activity relationships are discussed. Together, these data present a novel series of thiazolyl hydrazones with potential effects against Chagas disease and they could be important leads in continuing development against Chagas disease.