Strategies in Medicinal Chemistry to Discover New Hit Compounds against Ebola Virus: Challenges and Perspectives in Drug Discovery.

Ebola Virus (EBOV) is an infectious disease that mainly affects the cardiovascular system. It belongs to the Filoviridae family, consisting of filamentous envelopes and non-segmented negative RNA genome. EBOV was initially identified in Sudan and Zaire (now named the Democratic Republic of Congo) around 1967. It is transmitted mainly by contact with secretions (blood, sweat, saliva, and tears) from infected wild animals, such as non-human primates and bats. It has gained more prominence in recent years due to the recent EBOV outbreaks that occurred from 2013 to 2016, resulting in approximately 28,000 infected individuals, with a mortality rate of 40- 70%, affecting mainly Liberia, Guinea, and Sierra Leone. Despite these alarming levels, there is still no FDA-approved drug for the effective treatment of these diseases. The most advanced drug to treat EBOV is remdesivir. However, it is a high-cost drug and is available only for intravenous use. In this sense, more investments are needed in the research focused on the development of new antiviral drugs. In this context, medicinal chemistry strategies have been improving and increasingly discovering new hits that can be used in the future as a treatment against these diseases. Thus, this review will address the main advances in medicinal chemistry, such as drug discovery through computational techniques (virtual screening and virtual high throughput screening), drug repurposing, phenotypic screening assays, and employing classical medicinal chemistry, such as bioisosterism, metabolism-based drug design, and the discovery of new inhibitors through natural products, thereby presenting several promising compounds that may contain the advance of these pathogens.

Association between frailty phenotype, quantification of plasma HIV-1 RNA, CD4 cell count and HAART in HIV-positive subjects: a systematic review and meta-analysis of observational studies.

HIV infection causes a constant activation of the immune system and contributes to an enhanced systemic pro-inflammatory cytokine milieu, which has been associated with premature aging and frailty. We performed a systematic review and meta-analysis to analyze whether the HIV-1 RNA load, CD4+ T-lymphocyte counts and exposure to HAART in HIV-positive subjects are associated with frailty phenotype. Searches were performed in PubMed, SCOPUS, Lilacs, Web of Science, Google Scholar, and OpenThesis databases. We used the odds ratio as a measure of the association. We used either a fixed or random-effects model to pool the results of individual studies depending on the presence of heterogeneity. Eleven studies were included in the review. Data from 8035 HIV-positive subjects were analyzed; 2413 of the subjects had viral load detectable, 981 had a CD4T-cell count <350 cells/µL, and 1342 had HAART exposure information. We found an association between frailty and CD4T-cell count <350 cells/µL (OR 2.68, CI 95% 1.68-4.26, I2 = 46%), HIV-1 RNA load detectable (OR 1.71, CI 95% 1.38-2.12, I2 = 0%), and protease inhibitor-containing HAART regimen (OR 2.21, CI 95% 1.26-3.89, I2 = 0%). Further studies are necessary to evaluate the effects of other factors on the development of clinical features related to frailty.

Control of postharvest fungal diseases in fruits using external application of RNAi.

Contamination with a variety of filamentous fungi can cause deterioration of food and agricultural products. Fungal contaminations reduce the quality and the shelf life of fresh fruits and are one of the main causes of economic loss in the global fresh fruit industry. Although chemical fungicides are effective and traditionally used to control postharvest fungal diseases, they are harmful to human health. In this context, use of RNA interference (RNAi)-based fungicides is a promising alternative strategy. Spray-induced gene silencing (SIGS) is an innovative RNAi-based approach for silencing target genes in phytopathogens. This review aims to discuss the recent findings on the use of RNAi-based fungicides to control the postharvest spoilage of fresh fruits. PRACTICAL APPLICATION: Control of postharvest fungal diseases is one of the most important strategies to make food available to consumers longer. In this sense, the external application of RNAi seems to be technologically advantageous and efficient as it helps to maintain the characteristics of plant products. In this sense, this review discussed what is possible to find in the literature regarding this new technology.

Comportamiento virológico de pacientes con hepatitis C tratados con antivirales cubanos / Virological behavior of patients with hepatitis C treated with Cuban antivirals

Introducción: el seguimiento virológico de los pacientes con hepatitis C se realiza mediante la determinación cuantitativa del ácido ribonucleico viral por técnicas de biología molecular. Objetivos: evaluar el comportamiento virológico de los pacientes con hepatitis crónica C tratados con antivirales cubanos. Materiales y métodos: se realizó un estudio descriptivo, prospectivo en 45 pacientes con hepatitis crónica C atendidos en Consulta de Hepatología del Hospital Universitario Faustino Pérez, de Matanzas, en el período comprendido entre enero 2014 a diciembre 2017, tratados durante 48 semanas con PEG-heberon y ribavirina. De ellos se analizaron las características basales así como los diferentes tipos de respuesta al tratamiento según resultados virológicos. Resultados: predominaron los pacientes del sexo femenino, menores de 45 años, vírgenes de tratamiento y con cargas virales basales altas. Se alcanzó la respuesta virológica rápida en el 31,1%, la temprana total en el 19,4 %, al final del tratamiento en el 77,1% y la respuesta virológica sostenida en el 59,3%. Entre los respondedores predominaron los rápidos con respuesta virológica sostenida y entre los no respondedores, los nulos. Conclusiones: los estudios cuantitativos de ácido ribonucleico viral son esenciales para el seguimiento de los pacientes con hepatitis C ya que a través de sus determinaciones basales, durante el tratamiento y posterior a este, puede evaluarse la respuesta al tratamiento (AU).

Introduction: the virological follow-up of patients with hepatitis C is made through the quantitative determination of the viral ribonucleic acid using techniques of molecular biology. Objectives: to assess the virological behavior of patients with hepatitis C treated with Cuban antivirals. Materials and methods: a prospective, descriptive study was carried out in 45 patients with hepatitis C who attended the Consultation of Hepatology of the University Hospital "Faustino Pérez", of Matanzas, in the period from January 2014 to December 2017, treated with PEG-eberon and ribavirin for 48 weeks. Their basal characteristics were analyzed and also the different kinds of answer to the treatment according to the virological results. Results: female sex, patients aged less than 45 years old, non-treated before and with high viral loads. The fast virological answer was reached in 31 % of the patients, and the total early answer in 19.4 %; at the end of the treatment in 77.1 %, and the sustained virological answer in 59.3 % of the patients. Among the answering ones predominated the fast with sustained virological answer, and among the non-answering predominated the null ones. Conclusions: quantitative studies of viral ribonucleic acid are essential for the follow-up of patients with hepatitis C, because through their basal determinations, during and after the treatment, the answer to the treatment can be evaluated (AU).

Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica.

Messenger RNA 3'-end polyadenylation is an important regulator of gene expression in eukaryotic cells. In our search for new ways of treating parasitic infectious diseases, we looked at whether or not alterations in polyadenylation might control the survival of Entamoeba histolytica (the agent of amoebiasis in humans). We used molecular biology and computational tools to characterize the mRNA cleavage factor EhCFIm25, which is essential for polyadenylation in E. histolytica. By using a strategy based on the systematic evolution of ligands by exponential enrichment, we identified single-stranded RNA aptamers that target EhCFIm25. The results of RNA-protein binding assays showed that EhCFIm25 binds to the GUUG motif in vitro, which differs from the UGUA motif bound by the homologous human protein. Accordingly, docking experiments and molecular dynamic simulations confirmed that interaction with GUUG stabilizes EhCFIm25. Incubating E. histolytica trophozoites with selected aptamers inhibited parasite proliferation and rapidly led to cell death. Overall, our data indicate that targeting EhCFIm25 is an effective way of limiting the growth of E. histolytica in vitro. The present study is the first to have highlighted the potential value of RNA aptamers for controlling this human pathogen.

Regulatory RNA induces the production of IFN-gamma, but not IL-4 in human lymphocytes: role of RNA-dependent protein kinase (PKR) and NF-kappaB.

Previous results with p9-RNA, obtained from lymph nodes of animals immunized with the peptide p9 of HIV-1, suggested that its effects on lymphocytes could be mediated by RNA-dependent protein kinase (PKR). Here we report that p9-RNA activates PKR leading to the degradation of the inhibitor I-kappaB alpha and the concomitant nuclear factor kappa B (NF-kappaB) activation. The fractionation of p9-RNA by affinity chromatography indicates that the poly A(+) p9-RNA is the fraction responsible for PKR activation. We also found that p9-RNA induces the production of interferon-gamma (IFN-gamma), but not interleukin (IL-4) since only IFN-gamma gene promoter contains NF-kappaB binding site. This study provides the first evidence that transcriptional control of gene expression by regulatory RNAs can be mediated by PKR through NF-kappaB activation. A model for the mechanism of action of poly A(+) p9-RNA is proposed.

A point mutation in bioactive RNA results in the failure of mutant heart correction in Mexican axolotls.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. The defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or endoderm/mesoderm-conditioned medium. By screening a cDNA library made of total conditioned medium RNA from normal axolotl embryonic endoderm, we isolated a single clone (MIR), the synthetic RNA from which corrects the mutant heart defect by promoting myofibrillogenesis and thus was named MIR (myofibrillogenesis inducing RNA). In the present study, we have examined MIR gene expression in mutant axolotl hearts at early pre-heart-beat developmental stages and found its quantitative expression, as detected by RT-PCR, to be the same as in normal hearts. However, careful analysis of sequence data revealed a G-->U point mutation in the mutant MIR RNA. Further computational analyses, using GENEBEE software to compare normal and mutant MIR RNAs show a significant alteration in RNA secondary structure of the point-mutated MIR RNA. The results from bioassay and confocal microscopy immunofluorescent studies demonstrate that, unlike MIR RNA derived from normal embryos, the mutated MIR RNA does not promote myofibrillogenesis in mutant embryonic hearts and fails to rescue/correct the mutant heart defect.

A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. However, the defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or RNA from endoderm mesoderm-conditioned medium. We constructed a cDNA library from total conditioned medium RNA in a pcDNAII expression vector. We screened the cDNA library by an organ culture bioassay and isolated a single clone (Cl#4), the synthetic RNA from which corrects the heart defect by promoting myofibrillogenesis. The insert size of the active clone is 166 nt in length with a unique nucleotide sequence. The anti-sense RNA from Cl#4 using SP6 RNA polymerase failed to rescue mutant hearts. The ability of this small RNA to correct the mutant heart defect suggests that the RNA probably does not act as an mRNA. While the precise mechanism of action is not yet known, on the basis of our studies to date it is very clear that the sense strand of Cl#4 RNA has the ability to promote myofibrillogenesis and rescue the mutant hearts both in vitro and in vivo.

Effect of RNA on the immunological response of rats, induced by Salmonella typhimurium.

Rats treated with RNA from normal rat spleen were challenged 5 days later with Salmonella typhimurium. The primary response was delayed and antibody titers significantly lower than controls were observed. Forty eight hours after the secondary challenge the antibody titers were also lower; this difference disappeared five days later. This study confirms the immunological depression produced by RNA. Moreover, it is demonstrated that the RNA action is transitory and that the recovery of secondary response occurs seven days after the second challenge. This action is probably explained at macrophage level.