Abstract The disease coronavirus COVID-19 has been the cause of millions of deaths worldwide. Among the proteins of SARS-CoV-2, non-structural protein 12 (NSP12) plays a key role during COVID infection and is part of the RNA-dependent RNA polymerase complex. The monitoring of NSP12 polymorphisms is extremely important for the design of new antiviral drugs and monitoring of viral evolution. This study analyzed the NSP12 mutations detected in circulating SARS-CoV-2 during the years 2020 to 2022 in the population of the city of Manaus, Amazonas, Brazil. The most frequent mutations found were P323L and G671S. Reports in the literature indicate that these mutations are related to transmissibility efficiency, which may have contributed to the extremely high numbers of cases in this location. In addition, two mutations described here (E796D and R914K) are close and have RMSD that is similar to the mutations M794V and N911K, which have been described in the literature as influential on the performance of the NSP12 enzyme. These data demonstrate the need to monitor the emergence of new mutations in NSP12 in order to better understand their consequences for the treatments currently used and in the design of new drugs. COVID19 19 COVID-1 worldwide SARSCoV2, SARSCoV2 SARSCoV SARS CoV 2, 2 nonstructural non structural 1 NSP (NSP12 RNAdependent dependent complex NSP1 evolution SARS-CoV- 202 Manaus Amazonas Brazil PL P L G671S GS G S efficiency location addition E796D ED E D R914K RK R K MV M V N911K NK N enzyme COVID1 COVID- (NSP1 SARS-CoV 20 (NSP

Abstract Plasmodium falciparum is known to cause severe malaria, current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. Knowledge concerning P. falciparum essential proteins can be used for searching new antimalarials, among these a potential candidate is shikimate dehydrogenase (SDH), an enzyme part of the shikimate pathway which is responsible for producing endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unexplored by the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct an optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). As a result, optimal expression conditions were acquired by varying IPTG and temperature through time. Western Blot analysis was applied to verify appropriate PfSDH expression, solubilization and purification started with lysis followed by two-steps IMAC purification. Enzyme activity was measured spectrophotometrically by NADPH oxidation, optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours growing at 37 °C and shaking at 200 rpm, recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form. malaria artemisininbased artemisinin based therapy failure P antimalarials SDH, , (SDH) acids (PfSDH community therefore pET28aPfSDH pETaPfSDH pET28a pET E BL21DE3. BL21DE3 BLDE BL21 DE3 . BL DE BL21(DE3) result time twosteps two steps oxidation 01 0 1 0. 4 3 C 20 rpm confirmed Thus form (SDH aPfSDH pETa BL21DE BL2 BL21(DE3 2 BL21(DE

Malaria is responsible for thousands of deaths each year. Currently, artemisinin combination therapy (ACT) is used as first-choice medication against the disease. However, the emergence of resistant strains prompts the search for alternative compounds. The present study aimed to investigate the antiplasmodial activities of natural triterpenes (compounds 1 and 2), and semisynthetic derivatives 1a, 1b, 1c, and 1d. Antiplasmodial assays were carried out using the SYBR Green technique, whereas cytotoxicity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Hemolytic assays were performed on human erythrocytes. An in silico analysis of the compounds against PfENR (Plasmodium falciparum 2-trans-enoyl-reductase) was carried out by molecular docking. Experiments with 1, and its derivatives against P. falciparum showed that 1a was very similar in terms of biological activity to compound 1 (half maximal inhibitory concentration (IC50) ca. 4 µM), whereas 1b, 1c, and 1d had reduced antiplasmodial activities (IC50 between 8-103 µM). The selectivity indexes of 1 and 1d for HepG2, and Vero cells were > 10. Docking results partially agreed with the in vitro experiments, with 1 and 1c having the best and worst affinities with PfENR, respectively. In conclusion, the results showed that 1 and 1d may serve as biotechnological tools in the development of antimalarial drugs.