Synthesis and characterization of two known and one new impurities of dolutegravir: In silico evaluation of certain intermediates against SARS CoV-2 O-ribose methyltransferase (OMTase).

Besides its use against HIV infection the marketed anti-retroviral drug dolutegravir attracted attention as a potential agent against COVID-19 in multiple AI (artificial intelligence) based studies. Due to our interest in accessing the impurities of this drug we report the synthesis and characterization of three impurities of dolutegravir one of which is new. The synthesis of O-methyl ent-dolutegravir was accomplished in three-steps the first one involved the construction of fused 1,3-oxazinane ring. The cleavage of -OEt ether moiety followed by methylation afforded the target compound. The second impurity i.e. N-(2,4-difluorobenzyl)-4-methoxy-3-oxobutanamide was synthesized via a multi-step method involving sequentially the keto group protection, ester hydrolysis, acid chloride formation followed by the reaction with amine and finally keto group deprotection. The synthesis of new or dimer impurity was carried out via another multi-step method similar to the previous one starting from ethyl 4-chloro acetoacetate. The methodology involved preparation of ether derivative, keto group protection, ester hydrolysis, preparation of amide derivative via acid chloride formation in situ and then keto group deprotection for a longer duration. The last step afforded the target compound for which a plausible reaction mechanism has been proposed. All three impurities were prepared in gram scale (minimum 2 g and maximum 8 g). The in silico evaluation of three selected synthesized intermediates e.g. 7, 8 and 9 (structurally similar to dolutegravir) against SARS CoV-2 O-ribose methyltransferase (OMTase) (PDB: 3R24) indicated that compound 7 could be of interest as a possible inhibitor of this protein.

Molecular characteristics of SARS-CoV-2 Nsp16 protein and analysis of its potential effect on male reproductive function

Objective The possibility of coronavirus disease 2019 (COVID-19) involving injury to reproductive function has attracted attention. This study analyzed the genetic characteristics, molecular structure and biological function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp16 protein, and explored potential effects of Nsp16 on germ cells following the virus′ invading testicular tissue, aiming to lay basis for studies of pathogenic mechanisms and therapeutic strategies. Methods Bioinformatic techniques and international biological databases were used to analyze nsp16 genetic variability, Nsp16 spatial structure and function, and effects on genes and proteins of germ cells. DrugBank databases were applied in screening for drugs targeted at Nsp16. Results An evolutionary tree was constructed based on the nsp16 sequences of 30 isolates of 3 coronavirus species. The nsp16 conserved property was 99% amongst SARS-CoV-2 isolates. Nsp16 is a hydrophilic protein, with a 1.9 h half-life inside cells in vitro. Nsp16 has methyltransferase activity, showing potential to regulate gene and functional protein methylation of sperm and Leydig cells. Nsp16 has both linear B cell epitopes and CTL cell epitopes, with capacity to induce immune responses and damage to testicular tissue. Two inhibitory drugs targeted at Nsp16 were found by screening the DrugBank database. Conclusions SARS-CoV-2 Nsp16 is a functional protein encoded by a highly conserved gene, may affect germ cell growth and development by promoting methylation of host cellular genes and proteins following the virus′ invasion into testis tissue through angiotensin-converting enzyme 2 receptors. This report presents Nsp16-targeted chemotherapeutic drugs for the first time, showing high reference value for prevention and treatment of COVID-19 and related lesions of the male reproductive system. © 2021, Publication Centre of Anhui Medical University. All rights reserved.

Targeting SARS-CoV-2: a systematic drug repurposing approach to identify promising inhibitors against 3C-like proteinase and 2'-O-ribose methyltransferase.

The recent pandemic associated with SARS-CoV-2, a virus of the Coronaviridae family, has resulted in an unprecedented number of infected people. The highly contagious nature of this virus makes it imperative for us to identify promising inhibitors from pre-existing antiviral drugs. Two druggable targets, namely 3C-like proteinase (3CLpro) and 2'-O-ribose methyltransferase (2'-O-MTase) were selected in this study due to their indispensable nature in the viral life cycle. 3CLpro is a cysteine protease responsible for the proteolysis of replicase polyproteins resulting in the formation of various functional proteins, whereas 2'-O-MTase methylates the ribose 2'-O position of the first and second nucleotide of viral mRNA, which sequesters it from the host immune system. The selected drug target proteins were screened against an in-house library of 123 antiviral drugs. Two promising drug molecules were identified for each protein based on their estimated free energy of binding (ΔG), the orientation of drug molecules in the active site and the interacting residues. The selected protein-drug complexes were then subjected to MD simulation, which consists of various structural parameters to equivalently reflect their physiological state. From the virtual screening results, two drug molecules were selected for each drug target protein [Paritaprevir (ΔG = -9.8 kcal/mol) & Raltegravir (ΔG = -7.8 kcal/mol) for 3CLpro and Dolutegravir (ΔG = -9.4 kcal/mol) and Bictegravir (ΔG = -8.4 kcal/mol) for 2'-OMTase]. After the extensive computational analysis, we proposed that Raltegravir, Paritaprevir, Bictegravir and Dolutegravir are excellent lead candidates for these crucial proteins and they could become potential therapeutic drugs against SARS-CoV-2. Communicated by Ramaswamy H. Sarma.