Structural basis of nirmatrelvir and ensitrelvir activity against naturally occurring polymorphisms of the SARS-CoV-2 main protease.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.

Effects of Dimerization, Dendrimerization, and Chirality in p-BthTX-I Peptide Analogs on the Antibacterial Activity and Enzymatic Inhibition of the SARS-CoV-2 PLpro Protein.

Recent studies have shown that the peptide [des-Cys11,Lys12,Lys13-(p-BthTX-I)2K] (p-Bth) is a p-BthTX-I analog that shows enhanced antimicrobial activity, stability and hemolytic activity, and is easy to obtain compared to the wild-type sequence. This molecule also inhibits SARS-CoV-2 viral infection in Vero cells, acting on SARS-CoV-2 PLpro enzymatic activity. Thus, the present study aimed to assess the effects of structural modifications to p-Bth, such as dimerization, dendrimerization and chirality, on the antibacterial activity and inhibitory properties of PLpro. The results showed that the dimerization or dendrimerization of p-Bth was essential for antibacterial activity, as the monomeric structure led to a total loss of, or significant reduction in, bacterial activities. The dimers and tetramers obtained using branched lysine proved to be prominent compounds with antibacterial activity against Gram-positive and Gram-negative bacteria. In addition, hemolysis rates were below 10% at the corresponding concentrations. Conversely, the inhibitory activity of the PLpro of SARS-CoV-2 was similar in the monomeric, dimeric and tetrameric forms of p-Bth. Our findings indicate the importance of the dimerization and dendrimerization of this important class of antimicrobial peptides, which shows great potential for antimicrobial and antiviral drug-discovery campaigns.

Natural Compounds as Non-Nucleoside Inhibitors of Zika Virus Polymerase through Integration of In Silico and In Vitro Approaches.

Although the past epidemic of Zika virus (ZIKV) resulted in severe neurological consequences for infected infants and adults, there are still no approved drugs to treat ZIKV infection. In this study, we applied computational approaches to screen an in-house database of 77 natural and semi-synthetic compounds against ZIKV NS5 RNA-dependent RNA-polymerase (NS5 RdRp), an essential protein for viral RNA elongation during the replication process. For this purpose, we integrated computational approaches such as binding-site conservation, chemical space analysis and molecular docking. As a result, we prioritized nine virtual hits for experimental evaluation. Enzymatic assays confirmed that pedalitin and quercetin inhibited ZIKV NS5 RdRp with IC50 values of 4.1 and 0.5 µM, respectively. Moreover, pedalitin also displayed antiviral activity on ZIKV infection with an EC50 of 19.28 µM cell-based assays, with low toxicity in Vero cells (CC50 = 83.66 µM) and selectivity index of 4.34. These results demonstrate the potential of the natural compounds pedalitin and quercetin as candidates for structural optimization studies towards the discovery of new anti-ZIKV drug candidates.

Synthesis and antiplasmodial assessment of nitazoxanide and analogs as new antimalarial candidates.

During the last years, the progression to control malaria disease seems to be slowed and WHO (World Health Organization) reported a modeling analysis with the prediction of the increase in malaria morbidity and mortality in sub-Saharan Africa during the COVID-19 pandemic. A rapid way to the discovery of new drugs could be carried out by performing investigations to identify drugs based on repurposing of "old" drugs. The 5-nitrothiazole drug, Nitazoxanide was shown to be active against intestinal protozoa, human helminths, anaerobic bacteria, viruses, etc. In this work, Nitazoxanide and analogs were prepared using two methodologies and evaluated against P. falciparum 3D7. A bithiazole analog, showed attractive inhibitory activity with an EC50 value of 5.9 µM, low propensity to show toxic effect against HepG2 cells at 25 µM, and no cross-resistance with standard antimalarials.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum.

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

Anthelmintic activity of a nanoformulation based on thiophenes identified in Tagetes patula L. (Asteraceae) against the small ruminant nematode Haemonchus contortus.

The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5'-(hydroxymethyl)-[2,2'-bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting EC50 = 0.1731 mg.mL-1 and EC50 = 0.3243 mg.mL-1, respectively. So, this compound was selected to preparation of a nanostructured formulation to be orally administered to Santa Inês sheep. In general, from the fecal egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPGmean = 2167.1, efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthelmintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.mol-1) and the proposed binding mode, Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic assays.

Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies.

BACKGROUND: Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise. METHODS: The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed. RESULTS: A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively). CONCLUSIONS: The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

Discovery of Marinoquinolines as Potent and Fast-Acting Plasmodium falciparum Inhibitors with in Vivo Activity.

We report the discovery of marinoquinoline (3 H-pyrrolo[2,3- c]quinoline) derivatives as new chemotypes with antiplasmodial activity. We evaluated their inhibitory activities against P. falciparum and conducted a structure-activity relationship study, focusing on improving their potency and maintaining low cytotoxicity. Next, we devised quantitative structure-activity relationship (QSAR) models, which we prospectively validated, to discover new analogues with enhanced potency. The most potent compound, 50 (IC503d7 = 39 nM; IC50K1 = 41 nM), is a fast-acting inhibitor with dual-stage (blood and liver) activity. The compound showed considerable selectivity (SI > 6410), an additive effect when administered in combination with artesunate, excellent tolerability in mice (all mice survived after an oral treatment with a 1000 mg/kg dose), and oral efficacy at 50 mg/kg in a mouse model of P. berghei malaria (62% reduction in parasitemia on day 5 postinfection); thus, compound 50 was considered a lead compound for the discovery of new antimalarial agents.

Ceratocystis cacaofunesta genome analysis reveals a large expansion of extracellular phosphatidylinositol-specific phospholipase-C genes (PI-PLC).

BACKGROUND: The Ceratocystis genus harbors a large number of phytopathogenic fungi that cause xylem parenchyma degradation and vascular destruction on a broad range of economically important plants. Ceratocystis cacaofunesta is a necrotrophic fungus responsible for lethal wilt disease in cacao. The aim of this work is to analyze the genome of C. cacaofunesta through a comparative approach with genomes of other Sordariomycetes in order to better understand the molecular basis of pathogenicity in the Ceratocystis genus. RESULTS: We present an analysis of the C. cacaofunesta genome focusing on secreted proteins that might constitute pathogenicity factors. Comparative genome analyses among five Ceratocystidaceae species and 23 other Sordariomycetes fungi showed a strong reduction in gene content of the Ceratocystis genus. However, some gene families displayed a remarkable expansion, in particular, the Phosphatidylinositol specific phospholipases-C (PI-PLC) family. Also, evolutionary rate calculations suggest that the evolution process of this family was guided by positive selection. Interestingly, among the 82 PI-PLCs genes identified in the C. cacaofunesta genome, 70 genes encoding extracellular PI-PLCs are grouped in eight small scaffolds surrounded by transposon fragments and scars that could be involved in the rapid evolution of the PI-PLC family. Experimental secretome using LC-MS/MS validated 24% (86 proteins) of the total predicted secretome (342 proteins), including four PI-PLCs and other important pathogenicity factors. CONCLUSION: Analysis of the Ceratocystis cacaofunesta genome provides evidence that PI-PLCs may play a role in pathogenicity. Subsequent functional studies will be aimed at evaluating this hypothesis. The observed genetic arsenals, together with the analysis of the PI-PLC family shown in this work, reveal significant differences in the Ceratocystis genome compared to the classical vascular fungi, Verticillium and Fusarium. Altogether, our analyses provide new insights into the evolution and the molecular basis of plant pathogenicity.

Phthalimide Derivatives with Bioactivity against Plasmodium falciparum: Synthesis, Evaluation, and Computational Studies Involving bc 1 Cytochrome Inhibition.

We describe herein the design and synthesis of N-phenyl phthalimide derivatives with inhibitory activities against Plasmodium falciparum (sensitive and resistant strains) in the low micromolar range and noticeable selectivity indices against human cells. The best inhibitor, 4-amino-2-(4-methoxyphenyl)isoindoline-1,3-dione (10), showed a slow-acting mechanism similar to that of atovaquone. Enzymatic assay indicated that 10 inhibited P. falciparum cytochrome bc 1 complex. Molecular docking studies suggested the binding mode of the best hit to Qo site of the cytochrome bc 1 complex. Our findings suggest that 10 is a promising candidate for hit-to-lead development.

Insecticidal activity of an essential oil of Tagetes patula L. (Asteraceae) on common bed bug Cimex lectularius L. and molecular docking of major compounds at the catalytic site of ClAChE1.

Emerging resistance to insecticides has influenced pharmaceutical research and the search for alternatives to control the common bed bug Cimex lectularius. In this sense, natural products can play a major role. Tagetes patula, popularly known as dwarf marigold, is a plant native to North America with biocide potential. The aim of this work was to evaluate the biological activity of T. patula essential oil (EO) against adult common bed bugs via exposure to dry residues by the Impregnated Paper Disk Test (IPDT) using cypermethrin as a positive control. We selected the enzyme acetylcholinesterase as a target for modeling studies, with the intent of investigating the molecular basis of any biological activity of the EO. Chemical analysis of the EO was performed using gas chromatography coupled to mass spectrometry (GC-MS). Additionally, oral and dermal acute toxicity tests were performed according to Organization for Economic Cooperation and Development (OECD) guidelines. The sulforhodamine B assay (SRB) was performed to verify the cytotoxicity of EO to HaCaT cells. The EO eliminated 100 % of the bed bugs at 100 mg mL-1 with an LC50 value of 15.85 mg mL-1. GC-MS analysis identified α-terpinolene, limonene, piperitenone, and piperitone as major components of the mixture. Molecular modeling studies of these major compounds suggested that they are acetylcholinesterase inhibitors with good steric and electronic complementarity. The in vitro cytotoxicity evaluation revealed a LC50 = 37.06 µg mL-1 and in vivo acute toxicity showed an LC50 >4000 mg kg-1, indicating that the EO presents low risk of toxic side effects in humans. The T. patula essential oil components provide a promising strategy for controlling bed bug populations with low mammalian toxicity. These findings pave the way for further in vivo studies aimed at developing a safe and effective insecticide.

Synthetic chalcones and sulfonamides as new classes of Yersinia enterocolitica YopH tyrosine phosphatase inhibitors.

YopH plays a relevant role in three pathogenic species of Yersinia. Due to its importance in the prevention of the inflammatory response of the host, this enzyme has become a valid target for the identification and development of new inhibitors. In this work, an in-house library of 283 synthetic compounds was assayed against recombinant YopH from Yersinia enterocolitica. From these, four chalcone derivatives and one sulfonamide were identified for the first time as competitive inhibitors of YopH with binding affinity in the low micromolar range. Molecular modeling investigations indicated that the new inhibitors showed similar binding modes, establishing polar and hydrophobic contacts with key residues of the YopH binding site.

Molecular features for antitrypanosomal activity of thiosemicarbazones revealed by OPS-PLS QSAR studies.

A quantitative structure-activity relationship analysis was employed to explore the relationship between the molecular structure of thiosemicarbazone analogues and the inhibition of the cysteine protease cruzain, a validated target for Chagas' disease treatment. A data set containing 53 thiosemicarbazone derivatives was used to produce a quantitative model for activity prediction of unknown compounds. Several electronic descriptors were obtained through DFT calculations, along with a large amount of Dragon descriptors. The ordered predictor selection (OPS) algorithm was employed to select the most relevant descriptors to perform PLS regressions. With this procedure, significant correlation coefficients (r(2) = 0.85, q(2) = 0.78) were achieved. Furthermore, predicted values for an external test set are in good agreement with the experimental results, indicating the potential of the model for untested compounds. Additional validation tests were carried out, indicating that a robust and reliable model was obtained to be used in the design of new thiosemicarbazones with improved cruzain inhibition potential.

Synthesis, biological evaluation, and molecular modeling of chalcone derivatives as potent inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatases (PtpA and PtpB).

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure-activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC(50) values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

Advances in prodrug design.

The background of prodrug design is presented herein as the basis for introducing new and advanced latent systems, taking into account mainly the versatility of polymers and other macromolecules as carriers. PDEPT (Polymer-Directed Enzyme Prodrug Therapy); PELT (Polymer-Enzyme Liposome Therapy); CDS (Chemical Delivery System); ADEPT(Antibody-Directed Enzyme Prodrug Therapy); GDEPT/VDEPT (Gene-Directed Enzyme Prodrug Therapy/Virus-Directed Enzyme Prodrug Therapy); ODDS (Osteotropic Drug Delivery System) and LEAPT (Lectin-directed enzyme-activated prodrug therapy) are briefly described and some examples are given.

Synthesis and in vitro evaluation of potential antichagasic hydroxymethylnitrofurazone (NFOH-121): a new nitrofurazone prodrug.

The synthesis of mutual prodrugs of nitrofurazone with primaquine, using specific and nonspecific spacer groups, has been previously attempted seeking selective antichagasic agents. The intermediate reaction product, hydroxymethylnitrofurazone (NFOH-121), was isolated and tested in LLC-MK(2) culture cells infected with trypomastigotes forms of Trypanosoma cruzi showing higher trypanocidal activity than nitrofurazone and benznidazol in all stages. The mutagenicity tests showed that the prodrug was less toxic than the parent drug. Degradation assays were carried out in pH 1.2 and 7.4.

Diminuiçäo da atividade mutagênica do pró-fármaco NFOH-121 em relaçäo ao nitrofural (nitrofurazona) / Decreased mutagenic activity of the prodrug NFOH-121 related to nitrofurazona

A doença de Chagas representa um sério problema de saúde em pelo menos 17 países do continente americano. Cerca de 100 milhöes de pessoas vivem sob o risco de contrair doença e 16-18 milhöes já estäo infectadas. No Brasil, pelo menos 10 por cento das pessoas infectadas desenvolvem enfermidades cardíacas severas ou doenças digestivas crônicas. Os custos médicos para seu tratamento obrigatório podem atingir 250 milhöes de dólares por ano e somente dois fármacos estäo disponíveis: nifurtimox e benznidazol, ambos usados na fase aguda da doença. No Brasil, somente o benznidazol está disponível no mercado. Estudos recentes mostraram que o pró-fármaco NFOH-121 tem atividade antichagásica superior à da molécula matriz (nitrofurazona). No presente trabalho nós estudamos os efeitos tóxicos(mutagênicos) do pró-fármaco usando teste de Ames e cepas TA102 e TA98 de Salmonella typhimurium. Os resultados mostraram que a modificaçäo molecular efetuada na nitrofurazona(pró-fármaco NFOH-121 diminuíram a açäo mutagênica em 300-400 por cento. O composto NFOH-121 é um potencial fármaco antichagásico que deverá ser submetido aos testes pré-clínicos e clínicos.