A consensus reverse docking approach for identification of a competitive inhibitor of acetyltransferase enhanced intracellular survival protein from Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.

Larvicidal activity of diterpenes from Xylopia langsdorfiana St. Hilaire & Tulasne (Annonaceae) against Aedes aegypti linn. (diptera: culicidae).

Mosquitoes of the Aedes genus are responsible for transmitting many vector-borne viral diseases worldwide. Hundreds of thousands of people die annually from vector-borne diseases, including West Nile fever, dengue, tick-borne diseases, yellow fever, chikungunya, Rift Valley fever, and Zika. Billions of people are at the risk of infection on all continents, which is a cause of international concern. Therefore, new vector-control methods are essential for mitigating these illnesses. The bioactive hydrocarbons isolated from Xylopia langsdorfiana St. Hilaire & Tulasne are trachylobanes, a rare class of diterpenes found in the n-hexane fraction of the stem and leaf ethanolic extracts. These were tested against Ae. aegypti fourth-instar larvae over 48 h of exposure, with LC50 values ranging from 19.84 to 72.9 µg/mL, comparable to that of the positive control. The findings highlight the potential of Xylopia langsdorfiana St. Hilaire & Tulasne metabolites for controlling the main vectors of arthropod-borne viruses.

Design, synthesis, antiviral evaluation, and In silico studies of acrylamides targeting nsP2 from Chikungunya virus.

The Togaviridae family comprises several New- and Old-World Alphaviruses that have been responsible for thousands of human illnesses, including the RNA arbovirus Chikungunya virus (CHIKV). Firstly, it was reported in Tanzania in 1952 but rapidly it spread to several countries from Europe, Asia, and the Americas. Since then, CHIKV has been circulating in diverse countries around the world, leading to increased morbidity rates. Currently, there are no FDA-approved drugs or licensed vaccines to specifically treat CHIKV infections. Thus, there is a lack of alternatives to fight against this viral disease, making it an unmet need. Structurally, CHIKV is composed of five structural proteins (E3, E2, E1, C, and 6k) and four non-structural proteins (nsP1-4), in which nsP2 represents an attractive antiviral target for designing novel inhibitors since it has an essential role in the virus replication and transcription. Herein, we used a rational drug design strategy to select some acrylamide derivatives to be synthesized and evaluated against CHIKV nsP2 and also screened on CHIKV-infected cells. Thus, two regions of modifications were considered for these types of inhibitors, based on a previous study of our group, generating 1560 possible inhibitors. Then, the 24 most promising ones were synthesized and screened by using a FRET-based enzymatic assay protocol targeting CHIKV nsP2, identifying LQM330, 333, 336, and 338 as the most potent inhibitors, with Ki values of 48.6 ± 2.8, 92.3 ± 1.4, 2.3 ± 1.5, and 181.8 ± 2.5 µM, respectively. Still, their Km and Vmax kinetic parameters were also determined, along with their competitive binding modes of CHIKV nsP2 inhibition. Then, ITC analyses revealed KD values of 127, 159, 198, and 218 µM for LQM330, 333, 336, and 338, respectively. Also, their ΔH, ΔS, and ΔG physicochemical parameters were determined. MD simulations demonstrated that these inhibitors present a stable binding mode with nsP2, interacting with important residues of this protease, according to docking analyzes. Moreover, MM/PBSA calculations displayed that van der Waals interactions are mainly responsible for stabilizing the inhibitor-nsP2 complex, and their binding energies corroborated with their Ki values, having -198.7 ± 15.68, -124.8 ± 17.27, -247.4 ± 23.78, and -100.6 ± 19.21 kcal/mol for LQM330, 333, 336, and 338, respectively. Since Sindbis (SINV) nsP2 is similar to CHIKV nsP2, these best inhibitors were screened against SINV-infected cells, and it was verified that LQM330 presented the best result, with an EC50 value of 0.95 ± 0.09 µM. Even at 50 µM concentration, LQM338 was found to be cytotoxic on Vero cells after 48 h. Then, LQM330, 333, and 336 were evaluated against CHIKV-infected cells in antiviral assays, in which LQM330 was found to be the most promising antiviral candidate in this study, exhibiting an EC50 value of 5.2 ± 0.52 µM and SI of 31.78. The intracellular flow cytometry demonstrated that LQM330 is able to reduce the CHIKV cytopathogenic effect on cells, and also reduce the percentage of CHIKV-positive cells from 66.1% ± 7.05 to 35.8% ± 5.78 at 50 µM concentration. Finally, qPCR studies demonstrated that LQM330 was capable of reducing the number of viral RNA copies/µL, suggesting that CHIKV nsP2 is targeted by this inhibitor as its mechanism of action.

Targeting Chikungunya Virus Entry: Alternatives for New Inhibitors in Drug Discovery.

Chikungunya virus (CHIKV) is an Alphavirus (Togaviridae) responsible for Chikungunya fever (CHIKF) that is mainly characterized by a severe polyarthralgia, in which it is transmitted by the bite of infected Aedes aegypti and Ae. albopictus mosquitoes. Nowadays, there are no licensed vaccines or approved drugs to specifically treat this viral disease. Structural viral proteins participate in key steps of its replication cycle, such as viral entry, membrane fusion, nucleocapsid assembly, and virus budding. In this context, envelope E3-E2-E1 glycoproteins complex could be targeted for designing new drug candidates. In this review, aspects of the CHIKV entry mechanism are discussed to provide insights into assisting the drug discovery process. Moreover, several naturals, naturebased and synthetic compounds, as well as repurposed drugs and virtual screening are also explored as alternatives for developing CHIKV entry inhibitors. Finally, we provided a complementary analysis of studies involving inhibitors that were not explored by in silico methods. Based on this, Phe118, Val179, and Lys181 were found to be the most frequent residues, being present in 89.6, 82.7, and 93.1% of complexes, respectively. Lastly, some chemical aspects associated with interactions of these inhibitors and mature envelope E3- E2-E1 glycoproteins' complex were discussed to provide data for scientists worldwide, supporting their search for new inhibitors against this emerging arbovirus.

Insights on Dengue and Zika NS5 RNA-dependent RNA polymerase (RdRp) inhibitors.

Over recent years, many outbreaks caused by (re)emerging RNA viruses have been reported worldwide, including life-threatening Flaviviruses, such as Dengue (DENV) and Zika (ZIKV). Currently, there is only one licensed vaccine against Dengue, Dengvaxia®. However, its administration is not recommended for children under nine years. Still, there are no specific inhibitors available to treat these infectious diseases. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) is a metalloenzyme essential for viral replication, suggesting that it is a promising macromolecular target since it has no human homolog. Nowadays, several NS5 RdRp inhibitors have been reported, while none inhibitors are currently in clinical development. In this context, this review constitutes a comprehensive work focused on RdRp inhibitors from natural, synthetic, and even repurposing sources. Furthermore, their main aspects associated with the structure-activity relationship (SAR), proposed mechanisms of action, computational studies, and other topics will be discussed in detail.

Computer-Aided Design, Synthesis, and Antiviral Evaluation of Novel Acrylamides as Potential Inhibitors of E3-E2-E1 Glycoproteins Complex from Chikungunya Virus.

Chikungunya virus (CHIKV) causes an infectious disease characterized by inflammation and pain of the musculoskeletal tissues accompanied by swelling in the joints and cartilage damage. Currently, there are no licensed vaccines or chemotherapeutic agents to prevent or treat CHIKV infections. In this context, our research aimed to explore the potential in vitro anti-CHIKV activity of acrylamide derivatives. In silico methods were applied to 132 Michael's acceptors toward the six most important biological targets from CHIKV. Subsequently, the ten most promising acrylamides were selected and synthesized. From the cytotoxicity MTT assay, we verified that LQM330, 334, and 336 demonstrate high cell viability at 40 µM. Moreover, these derivatives exhibited anti-CHIKV activities, highlighting the compound LQM334 which exhibited an inhibition value of 81%. Thus, docking simulations were performed to suggest a potential CHIKV-target for LQM334. It was observed that the LQM334 has a high affinity towards the E3-E2-E1 glycoproteins complex. Moreover, LQM334 reduced the percentage of CHIKV-positive cells from 74.07 to 0.88%, 48h post-treatment on intracellular flow cytometry staining. In conclusion, all virtual simulations corroborated with experimental results, and LQM334 could be used as a promising anti-CHIKV scaffold for designing new drugs in the future.

Evaluation of the extract of Zeyheria tuberculosa with a view to products for wound healing.

OBJECTIVES: to evaluate the antimicrobial, cytotoxic and healing activities of the ethanolic extract of the stems of Z. tuberculosa via topical use and/or oral ingestion. METHOD: antimicrobial assays in vitro using the disk diffusion method, the Artemia salina toxicity test, and in vivo assays with Wistar rats. From these was collected clinical, histological and biochemical data for evaluating the healing process. RESULTS: in vitro antimicrobial testing showed activity in relation to Streptococcus pyogenes, Staphylococcus aureus and Staphylococcus epidermidis, with zones of inhibition of 18, 14 and 10 mm, respectively. The best minimum inhibitory concentration was 62.5 µg/ml for S. aureus, this bacteria being chosen for the in vitro assays. Animals treated with the ointments with the extract of Z. tuberculosa showed the best results in the reduction of the wound diameter, data confirmed by the presence of re-epithelialization in the histological samples. CONCLUSION: the extract was shown to be promising for the continuation of studies which may identify the active ingredients responsible for the pharmacological activity and its mechanism of action in the process of wound healing, so as to develop a product which may be used as an alternate means in the repair of infected cutaneous wounds.

Evaluation of the extract of Zeyheria tuberculosa with a view to products for wound healing / Avaliação do extrato da Zeyheria tuberculosa na perspectiva de um produto para cicatrização de feridas / Evaluación del extracto de la Zeyheria tuberculosa en la perspectiva de un producto para cicatrización de heridas

OBJECTIVES: to evaluate the antimicrobial, cytotoxic and healing activities of the ethanolic extract of the stems of Z. tuberculosa via topical use and/or oral ingestion. METHOD: antimicrobial assays in vitro using the disk diffusion method, the Artemia salina toxicity test, and in vivo assays with Wistar rats. From these was collected clinical, histological and biochemical data for evaluating the healing process. RESULTS: in vitro antimicrobial testing showed activity in relation to Streptococcus pyogenes, Staphylococcus aureus and Staphylococcus epidermidis, with zones of inhibition of 18, 14 and 10 mm, respectively. The best minimum inhibitory concentration was 62.5 µg/ml for S. aureus, this bacteria being chosen for the in vitro assays. Animals treated with the ointments with the extract of Z. tuberculosa showed the best results in the reduction of the wound diameter, data confirmed by the presence of re-epithelialization in the histological samples. CONCLUSION: the extract was shown to be promising for the continuation of studies which may identify the active ingredients responsible for the pharmacological activity and its mechanism of action in the process of wound healing, so as to develop a product which may be used as an alternate means in the repair of infected cutaneous wounds. .

OBJETIVOS: avaliar as atividades antimicrobiana, citotóxica e cicatrizante do extrato etanólico do caule da Z. tuberculosa por via tópica e/ou ingestão oral. MÉTODO: ensaios antimicrobianos in vitro pelo método de difusão em disco, teste de toxicidade da Artemia salina e ensaios in vivo com ratos Wistar. Nesses foram coletados dados clínicos, histológicos e bioquímicos para avaliação do processo de cicatrização. RESULTADOS: ensaios antimicrobianos in vitro mostraram atividade frente à Streptococcus pyogenes, Staphylococcus aureus e Staphylococcus epidermidis, com halos de inibição de 18, 14 e 10mm, respectivamente. A melhor concentração inibitória mínima foi 62,5µg/mL para S. aureus, sendo essa bactéria escolhida para os ensaios in vivo. Animais tratados com as pomadas do extrato da Z. tuberculosa apresentaram melhores resultados na redução do diâmetro da ferida, dado confirmado pela presença de reepitelização nos cortes histológicos. CONCLUSÃO: o extrato mostrou-se promissor para a continuação de estudos que identifiquem os princípios ativos responsáveis pela atividade farmacológica e seu mecanismo de ação no processo de cicatrização de feridas, a fim de desenvolver um produto que possa ser utilizado de forma alternativa no reparo de feridas cutâneas infectadas. .

OBJETIVOS: evaluar las actividades antimicrobianas, citotóxicas y cicatrizantes del extracto etanólico del tallo de la Z. tuberculosa por vía tópica y/o ingestión oral. MÉTODO: ensayos antimicrobianos in vitro por el método de difusión en disco, prueba de toxicidad de la Artemia salina y ensayos in vivo con ratones Wistar. En estos fueron recolectados datos clínicos, histológicos y bioquímicos para evaluación del proceso de cicatrización. RESULTADOS: los ensayos antimicrobianos in vitro mostraron actividad frente a la Streptococcus pyogenes, Staphylococcus aureus y Staphylococcus epidermidis, con halos de inhibición de 18, 14 y 10 mm, respectivamente. La mejor concentración inhibitoria mínima fue 62,5 µg/mL para S. aureus, siendo esta bacteria escogida para los ensayos in vivo. Animales tratados con las pomadas del extracto de la Z. tuberculosa presentaron mejores resultados en la reducción del diámetro de la herida, dato confirmado por la presencia de reepitelización en los cortes histológicos. CONCLUSIÓN: el extracto se mostró promisor para la continuación de estudios que identifiquen los principios activos responsables por la actividad farmacológica y su mecanismo de acción en el proceso de cicatrización de heridas, con la finalidad de desarrollar un producto que pueda ser utilizado de forma alternativa en la reparación de heridas cutáneas infectadas. .