Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

Leaf tissue metabolomics fingerprinting of Citronella gongonha Mart. by 1H HR-MAS NMR.

This research characterizes key metabolites in the leaf from Citronella gongonha Martius (Mart.) Howard (Cardiopteridaceae). All metabolites were assessed in intact leaf tissue by proton (1H) high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy integrated with the principal component analysis (PCA) to depict molecular association with the seasonal change. The major 'known unknown' metabolites detected in 1H HR-MAS NMR were derivatives of flavonoid, polyphenolic and monoterpenoid compounds such as kaempferol-3-O-dihexoside, caffeoyl glucoside (2), 3-O-caffeoylquinic acid (3), 5-O-caffeoylquinic acid (4), kingiside (5), 8-epi-kingisidic acid (6), (7α)-7-O-methylmorroniside (7), (7ß)-7-O-methylmorroniside (8) and alpigenoside (9) together with the universally occurring sucrose (10), α-glucoses (11, 12), alanine (13), and fatty (linolenic) acid (14). Several of the major metabolites (1, 2-9) were additionally confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). In regard with the PCA results, metabolites 1, 2-9 and 14 were influenced by seasonal variation and/or from further (a) biotic environmental conditions. The findings in this work indicate that C. gongonha Mart. is an effective medicinal plant by preserving particularly compounds 2, 3-9 in abundant amounts. Because of close susceptibility with seasonal shift and ecological trends, further longitudinal studies are needed to realize the physiology and mechanism involved in the production of these and new metabolites in this plant under controlled conditions. Also, future studies are recommended to classify different epimers, especially of the phenolics and monoterpenoids in the given plant.

Predicting the Relative Binding Affinity for Reversible Covalent Inhibitors by Free Energy Perturbation Calculations.

Covalent inhibitors are assuming central importance in drug discovery projects, especially in this pandemic scenario. Many research groups have focused their attention on inhibiting viral proteases or human proteases such as cathepsin L (hCatL). The inhibition of these critical enzymes may impair viral replication. However, molecular modeling of covalent ligands is challenging since covalent and noncovalent ligand-bound states must be considered in the binding process. In this work, we evaluated the suitability of free energy perturbation (FEP) calculations as a tool for predicting the binding affinity of reversible covalent inhibitors of hCatL. Our strategy relies on the relative free energy calculated for both covalent and noncovalent complexes and the free energy changes have been compared with experimental data for eight nitrile-based inhibitors, including three new inhibitors of hCatL. Our results demonstrate that the covalent complex can be employed to properly rank the inhibitors. Nevertheless, a comparison of the free energy changes in both noncovalent and covalent states is valuable to interpret the effect triggered by the formation of the covalent bond on the interactions played by functional groups distant from the warhead. Overall, FEP can be employed as a powerful predictor tool in developing and understanding the activity of reversible covalent inhibitors.

Antioxidant and anti-sickling activity of glucal-based triazoles compounds - An in vitro and in silico study.

The sickle cell disease (SCD) has a genetic cause, characterized by a replacement of glutamic acid to valine in the ß-chain of hemoglobin. The disease has no effective treatment so far, and patients suffer a range from acute to chronic complications that include chronic hemolytic anemia, vaso-occlusive ischemia, pain, acute thoracic syndrome, cerebrovascular accident, nephropathy, osteonecrosis and reduced lifetime. The oxidation in certain regions of the hemoglobin favors the reactive oxygen species (ROS) formation, which is the cause of many clinical manifestations. Antioxidants have been studied to reduce the hemoglobin ROS levels, and in this sense, we have searched for new antioxidants glucal-based triazoles compounds with anti-sickling activity. Thirty analogues were synthetized and tested in in vitro antioxidant assays. Two of them were selected based in their effects and concentration-response activity and conducted to in cell assays. Both molecules did not cause any hemolysis and could reduce the red blood cell damage caused by hydrogen peroxide, in a model of oxidative stress induction that mimics the SCD. Moreover, one molecule (termed 11m), besides reducing the hemolysis, was able to prevent the cell damage caused by the hydrogen peroxide. Later on, by in silico pharmacokinetics analysis, we could see that 11m has appropriated proprieties for druggability and the probable mechanism of action is the binding to Peroxiredoxin-5, an antioxidant enzyme that reduces the hydrogen peroxide levels, verified after molecular docking assays. Thus, starting from 30 glucal-based triazoles molecules in a structure-activity relationship, we could select one with antioxidant proprieties that could act on RBC to reduce the oxidative stress, being useful for the treatment of SCD.

Design, synthesis and stepwise optimization of nitrile-based inhibitors of cathepsins B and L.

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e â†’ 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins.

Synthesis, biochemical evaluation and molecular modeling studies of nonpeptidic nitrile-based fluorinated compounds.

Aim: Compounds that block enzyme activity can kill pathogens and help develop effective and safe drugs for Chagas disease and leishmaniasis. Materials & methods: A library of nonpeptidic nitrile-based compounds was synthesized and had their inhibitory affinity tested against cruzain, Leishmania mexicana cysteine protease B and cathepsin L. Isothermal titration calorimetry experiments and molecular simulations were performed for selected compounds to obtain thermodynamic fingerprints and identify main interactions and putative modes of binding with cruzain. Results: The derivatives provided increased affinity against all enzymes compared with the lead, and thermodynamic and computational studies showed improved thermodynamic properties and a possible different mode of binding. Conclusion: Our studies culminated in 1b, a compound 60-fold more potent in cruzain than its lead that also showed entropic and enthalpic contributions favorable to Gibbs binding energy.

Dipeptidyl nitrile derivatives suppress the Trypanosoma cruzi in vitro infection.

Chagas disease affects several countries around the world with health and sanitation problems. Cysteine proteases are essential for the virulence and replication of the Trypanosoma cruzi, being modulated by dipeptidyl nitriles and derivatives. Here, four dipeptidyl nitrile derivatives were assayed in three T. cruzi morphologies and two strains (Tulahuen and Y) using a set of assays: (i) analysis of the inhibitory activity against cysteine proteases; (ii) determination of the cytotoxic activity and selectivity index; (iii) verification of the inhibition of the trypomastigote invasion in the host cell. These compounds could inhibit the activity of cysteine proteases using the selective substrate Z-FR-MCA for the trypomastigote lysate and extracellular amastigotes. Interestingly, these compounds did not present relevant enzymatic inhibition for the epimastigote lysate. Most of the substances were also cytotoxic and selective against the trypomastigotes and intracellular amastigotes. The best compound of the series (Neq0662) could reduce the enzymatic activity of the cysteine proteases for the trypomastigotes and amastigotes. It was equipotent to the benznidazole drug in the cytotoxic studies using these two parasite forms. Neq0662 was also selective for the parasite, and it inhibited the invasion of the mammalian host cell in all conditions tested at 10 µM. The stereochemistry of the trifluoromethyl group was an important factor for the bioactivity when the two diastereomers (Neq0662 and Neq0663) were compared. All-in-all, these results indicate that these compounds could move further in the drug development stage because of its promising bioactive profile.

Optimization strategy of single-digit nanomolar cross-class inhibitors of mammalian and protozoa cysteine proteases.

Cysteine proteases (CPs) are involved in a myriad of actions that include not only protein degradation, but also play an essential biological role in infectious and systemic diseases such as cancer. CPs also act as biomarkers and can be reached by active-based probes for diagnostic and mechanistic purposes that are critical in health and disease. In this paper, we present the modulation of a CP panel of parasites and mammals (Trypanosoma cruzi cruzain, LmCPB, CatK, CatL and CatS), whose inhibition by nitrile peptidomimetics allowed the identification of specificity and selectivity for a given CP. The activity cliffs identified at the CP inhibition level are useful for retrieving trends through multiple structure-activity relationships. For two of the cruzain inhibitors (10g and 4e), both enthalpy and entropy are favourable to Gibbs binding energy, thus overcoming enthalpy-entropy compensation (EEC). Group contribution of individual molecular modification through changes in enthalpy and entropy results in a separate partition on the relative differences of Gibbs binding energy (ΔΔG). Overall, this study highlights the role of CPs in polypharmacology and multi-target screening, which represents an imperative trend in the actual drug discovery effort.

Stereoselective oxa-Michael addition of tyrosine to propargyl aldehyde/esters: formation of benzofurans and flavones

The steroselective oxa-Michael addition of the phenol moiety present in tyrosine and 3-iodotyrosine to different propargyl aldehydes delivered products with predominantly Z stereochemistry, as evidenced by X-ray crystallography analysis. When ethyl ropiolate was used as the propargyl ester source, the products were achieved with exclusively E stereochemistry with yields ranging from 17% to 91%. The oxa-Michael addition compounds served as substrates in the synthesis of 5- and 6-membered heterocyclic compounds. The atmosphere applied to the reaction medium directly influenced the formation of the products. When an inert atmosphere of nitrogen was applied, a 2-aryl-3-formyl-5-alanylbenzofuran core was selectively obtained via a Heck intramolecular reaction, while the reactions carried out under a carbon monoxide atmosphere led exclusively to 6-alanyl-2-arylflavone derivatives via reductive intramolecular acylation.

Stereoselective oxa-Michael addition of tyrosine to propargyl aldehyde/esters: formation of benzofurans and flavones

The steroselective oxa-Michael addition of the phenol moiety present in tyrosine and 3-iodotyrosine to different propargyl aldehydes delivered products with predominantly Z stereochemistry, as evidenced by X-ray crystallography analysis. When ethyl ropiolate was used as the propargyl ester source, the products were achieved with exclusively E stereochemistry with yields ranging from 17% to 91%. The oxa-Michael addition compounds served as substrates in the synthesis of 5- and 6-membered heterocyclic compounds. The atmosphere applied to the reaction medium directly influenced the formation of the products. When an inert atmosphere of nitrogen was applied, a 2-aryl-3-formyl-5-alanylbenzofuran core was selectively obtained via a Heck intramolecular reaction, while the reactions carried out under a carbon monoxide atmosphere led exclusively to 6-alanyl-2-arylflavone derivatives via reductive intramolecular acylation.

3-Alkenyltyrosines accessed by Suzuki-Miyaura coupling: a key intermediate in the synthesis and mechanistic study of povarov multicomponent reactions

3-Vinyl and 3-stilbenyl tyrosine derivatives, accessed by the Suzuki-Miyaura coupling reaction in good yields, are presented. These synthetic olefins then provided a fast and effective synthesis of functionalized quinolines by a silver-catalyzed, microwave-mediated Povarov reaction in just 40min. In addition, we demonstrate a tandem one-pot microwave-mediated Povarov-CuAAC (copper-catalyzed azide-alkyne cycloaddition) synthesis to provide a 1,2,3-triazole ring with 35% overall yield. We propose a Povarov/aromatization mechanism, based on high-resolution mass spectrometry experiments, combined with computational studies based on density functional theory (DFT) methods.

Functionalization of protected tyrosine via Sonogashira reaction: synthesis of 3-(1,2,3-triazolyl)-tyrosine.

1,2,3-Triazol tyrosines were synthesized from tyrosine alkynes that were in turn prepared via Sonogashira cross-coupling reaction. The tyrosine alkynes were subjected to click-chemistry reaction conditions leading to the corresponding 3-(1,2,3-triazolyl)-tyrosines in yields ranging from moderate to good.

Synthesis of enyne and aryl vinyl sulfoxides: functionalization via Pummerer rearrangement.

An efficient methodology for the synthesis of aryl-substituted vinyl sulfoxides through direct substitution of aryl-substituted alkynyl grignard reagents on menthyl-p-toluenesulfinate followed by Suzuki-Miyaura cross-coupling reaction has been developed. It has also been described that the reaction of alkyl-substituted and cycloalkyl-substituted alkynyl grignard reagents with menthyl-p-toluenesulfinate led to two products, i.e., alkynyl sulfoxide derivatives, as a result of substitution, and enyne sulfoxide derivatives, which resulted from substitution followed by Michael type addition. It was possible to selectively synthesize the enyne sulfoxide derivatives by changing the concentration of the grignard reagent. These alkenyl sulfoxides were transformed into the corresponding [Formula: see text]-thio aldehydes in high yields via additive Pummerer rearrangement.

Crystal structure of [(2R,3R,4S)-3,4-bis(acet-yloxy)-5-iodo-3,4-di-hydro-2H-pyran-2-yl]methyl acetate.

In the title compound, C12H15IO7, the 3,4-di-hydro-2H-pyran ring is in a distorted half-boat conformation with the atom bearing the acet-yloxy group adjacent to the C atom bearing the methyl-acetate group lying 0.633 (6) Šabove the plane of the remaining ring atoms (r.m.s. deviation = 0.0907 Å). In the crystal, mol-ecules are linked into a supra-molecular chain along the a axis through two C-H⋯O inter-actions to the same acceptor carbonyl O atom; these chains pack with no specific inter-molecular inter-actions between them.

Synthesis of C-glycosyl-bis-1,2,3-triazole derivatives from 3,4,6-tri-O-acetyl-D-glucal.

We have developed an efficient, CuI-catalyzed, microwave-assisted method for the synthesis of bis-1,2,3-triazole derivatives starting from a 3,4,6-tri-O-acetyl-D-glucal-derived mesylate. This mesylate was obtained from 3,4,6-tri-O-acetyl-D-glucal through C-glycosidation, deprotection of acetate groups to alcohols, and selective mesylation of the primary alcohol. This mesylate moiety was then converted to an azide through a microwave-assisted method with good yield. The azide, once synthesized, was then treated with different terminal alkynes in the presence of CuI to synthesize various bis-triazoles in high yields and short reaction times.