Integration of biophysical and biological approaches to validate fragment-like compounds targeting l,d-transpeptidases from Mycobacterium tuberculosis.

Tuberculosis is one of the major causes of death worldwide; more than a million people die every year because of this infection. The constant emergency of Mycobacterium tuberculosis resistant strains against the most used treatments also contributes to the burden caused by this disease. Consequently, the development of new alternative therapies against this disease is constantly required. In recent years, only a few molecules have reached the market as new antituberculosis agents. The mycobacterial cell wall biosynthesis is for a longstanding considered an important target for drug development. Particularly, in M. tuberculosis, the peptidoglycan cross-links are predominantly formed by nonclassical bridges between the third residues of adjacent tetrapeptides. The responsible enzymes for these reactions are ld-transpeptidases (Ldts), for which M. tuberculosis has five paralogues. Although these enzymes are distinct from the penicillin-binding proteins (PBPs), they can also be inactivated by ß-lactam antibiotics, but since M. tuberculosis has a chromosomal ß-lactamase, most of the antibiotics of these classes can be degraded. Thus, to identify alternative scaffolds for the development of new antimicrobials against tuberculosis, we have integrated several fragment-based drug discovery techniques. Based on that, we identified and validated a number of small molecules that could be the starting point in the synthesis of more potent inhibitors against at least two Ldts from M. tuberculosis, LdtMt2 and LdtMt3. Eight identified molecules inhibited the Ldts activity in at least 20%, and three of them have antimycobacterial activity. The cell ultrastructural analysis suggested that one of the best compounds induced severe effects on the septum and cell wall morphologies, which corroborates our target-based approach to identifying new Ldts hits.

The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection.

BACKGROUND: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. OBJECTIVE: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. METHODS: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. RESULTS: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 µM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. CONCLUSION: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase.

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

From Medicinal Chemistry to Human Health: Current Approaches to Drug Discovery for Cancer and Neglected Tropical Diseases

ABSTRACT Scientific and technological breakthroughs have compelled the current players in drug discovery to increasingly incorporate knowledge-based approaches. This evolving paradigm, which has its roots attached to the recent advances in medicinal chemistry, molecular and structural biology, has unprecedentedly demanded the development of up-to-date computational approaches, such as bio- and chemo-informatics. These tools have been pivotal to catalyzing the ever-increasing amount of data generated by the molecular sciences, and to converting the data into insightful guidelines for use in the research pipeline. As a result, ligand- and structure-based drug design have emerged as key pathways to address the pharmaceutical industry's striking demands for innovation. These approaches depend on a keen integration of experimental and molecular modeling methods to surmount the main challenges faced by drug candidates - in vivo efficacy, pharmacodynamics, metabolism, pharmacokinetics and safety. To that end, the Laboratório de Química Medicinal e Computacional (LQMC) of the Universidade de São Paulo has developed forefront research on highly prevalent and life-threatening neglected tropical diseases and cancer. By taking part in global initiatives for pharmaceutical innovation, the laboratory has contributed to the advance of these critical therapeutic areas through the use of cutting-edge strategies in medicinal chemistry.

Planejamento de fármacos, biotecnologia e química medicinal: aplicações em doenças infecciosas / Planning pharmaceuticals, biotechnology and medicinal chemistry: applications in infectious diseases

Estratégias modernas de planejamento de fármacos se fundamentam no conhecimento da fisiopatologia das doenças, no estudo de vias bioquímicas e na seleção de alvos moleculares. As ferramentas biotecnológicas modernas têm fornecido informações valiosas para a descoberta e o desenvolvimento de novos fármacos. A química medicinal possui papel central em vários processos que visam à identificação de substâncias bioativas e ao desenvolvimento de compostos-líderes com propriedades farmacodinâmicas e farmacocinéticas otimizadas. O presente artigo apresenta uma abordagem de alguns aspectos fundamentais da biotecnologia e da química medicinal como ferramentas úteis para o planejamento de candidatos a novos fármacos para a terapia de doenças infecciosas.

Current drug design strategies are based on the understanding of the physiopathology of diseases, biochemical pathways and selection of molecular targets. Modern biotechnological tools have provided valuable information to facilitate the discovery of new drug candidates. Medicinal chemistry has a vital role in a variety of processes aimed at the identification of bioactive substances and the development of lead-compounds with optimized pharmacodynamic and pharmacokinetic properties. The present paper presents some fundamental aspects of biotechnology and medicinal chemistry as useful tools in the design of new chemical entities for the therapy of infectious diseases.

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors / Busca de inibidores da enzima glicossomal gliceraldeído 3-fosfato desidrogenase de Trypanosoma cruzi

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA percent > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Nesse trabalho foi avaliada a atividade inibitória sobre a enzima glicossomal gliceraldeído-3-fosfato desidrogenase de T. cruzi (gGAPDH) de extratos vegetais oriundos de plantas das famílias Meliaceae e Rutaceae, na concentração de 100 μg/mL. Foram testados 46 extratos, dos quais 15 apresentaram atividade inibitória significativa ( por cento AI > 50). A maioria dos extratos de plantas da família Meliaceae (Cedrela fissilis, Cipadessa fruticosa e Trichilia ramalhoi) apresentou grande potencial em inibir a atividade enzimática. O fracionamento do extrato hexânico dos galhos de C. fruticosa permitiu o isolamento de três flavonóides: flavona, 7-metoxiflavona e 3',4',5',5,7-pentametoxiflavona. Os dois últimos foram ativos na inibição da atividade de gGAPDH. Desta forma, as três espécies de Meliaceae testadas podem ser consideradas promissoras na busca de compostos protótipos para o controle da doença de Chagas.

Antibiotic, cytotoxic and enzyme inhibitory activity of crude extracts from Brazilian marine invertebrates

Herein we present the results of a screening with 349 crude extracts of Brazilian marine sponges, ascidians, bryozoans and octocorals, against 16 strains of susceptible and antibiotic-resistant bacteria, one yeast (Candida albicans), Mycobacterium tuberculosis H37Rv, three cancer cell lines MCF-7 (breast), B16 (murine melanoma ) and HCT8 (colon), and Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT) enzyme. Less than 15 percent of marine sponge crude extracts displayed antibacterial activity, both against susceptible and antibiotic-resistant bacteria. Up to 40 percent of marine sponge crude extracts displayed antimycobacterial activity against M. tuberculosis H37Rv. Cytotoxicity was observed for 18 percent of marine sponge crude extracts. Finally, less than 3 percent of sponge extracts inhibited L-APRT. Less than 10 percent of ascidian crude extracts displayed antibacterial activity. More than 25 percent of ascidian crude extracts were active against M. tuberculosis and the three cancer cell lines. Only two crude extracts from the ascidian Polysyncraton sp. collected in different seasons (1995 and 1997) displayed activity against L-APRT. Less than 2 percent of bryozoan and octocoral crude extracts presented antibacterial activity, but a high percentage of crude extracts from bryozoan and octororal displayed cytotoxic (11 percent and 30 percent, respectively) and antimycobacterial (60 percent) activities. The extract of only one species of bryozoan, Bugula sp., presented inhibitory activity against L-APRT. Overall, the crude extracts of marine invertebrates herein investigated presented a high level of cytotoxic and antimycobacterial activities, a lower level of antibacterial activity and only a small number of crude extracts inhibited L-APRT. Taxonomic analysis of some of the more potently active crude extracts showed the occurrence of biological activity in taxa that have been previously chemically investigated. These...

No presente estudo apresentamos resultados da triagem biológica realizada com 349 extratos obtidos de esponjas marinhas, ascídias, briozoários e octocorais do Brasil, em testes contra 16 linhagens de bactérias comuns e resistentes à antibióticos, uma levedura (Candida albicans), Mycobacterium tuberculosis H37Rv, três linhagens de células tumorais MCF-7 (mama), B16 (melanoma murínico) e HCT8 (cólon), e de inibição da enzima adenina fosforribosil transferase de Leishmania tarentolae (L-APRT). Menos de 15 por cento dos extratos de esponja marinhas apresentaram atividade antibacteriana, contra linhagens resistentes ou não a antibióticos. Quase 40 por cento dos extratos de esponjas marinhas apresentaram atividade antimicobacteriana contra Mycobacterium tuberculosis H37Rv. Foi observada citotoxicidade para 18 por cento dos extratos de esponjas marinhas. Finalmente, menos de 3 por cento dos extratos de esponjas apresentaram atividade inibitória da enzima L-APRT. Menos de 10 por cento dos extratos de ascídias apresentaram atividade antibacteriana. Mais de 25 por cento dos extratos de ascídias apresentaram atividade contra M. tuberculosis e as três linhagens de células tumorais. Somente dois extratos obtidos da ascídia Polysyncraton sp. coletada em duas diferentes épocas (1995 e 1997) apresentaram atividade contra L-APRT. Menos de 2 por cento dos extratos de briozoários e octocorais apresentaram atividade antibacteriana, mas uma alta percentagem de extratos destes animais apresentaram atividades citotóxica (11 por cento e 30 por cento, respectivamente) e antimicobacteriana (60 por cento). O extrato de somente uma espécie de briozoário, Bugula sp., apresentou atividade inibitória da enzima L-APRT. A análise taxonômica de algumas espécies de invertebrados que forneceram alguns dos extratos mais ativos, indicou a ocorrência de atividade biológica em espécies pertencentes a grupos taxonômicos que já foram anteriormente investigados do ponto de vista...