Computational and Enzymatic Studies of Sartans in SARS-CoV-2 Spike RBD-ACE2 Binding: The Role of Tetrazole and Perspectives as Antihypertensive and COVID-19 Therapeutics.

This study is an extension of current research into a novel class of synthetic antihypertensive drugs referred to as "bisartans", which are bis-alkylated imidazole derivatives bearing two symmetric anionic biphenyltetrazoles. Research to date indicates that bisartans are superior to commercially available hypertension drugs, since the former undergo stronger docking to angiotensin-converting enzyme 2 (ACE2). ACE2 is the key receptor involved in SARS-CoV-2 entry, thus initiating COVID-19 infection and in regulating levels of vasoactive peptides such as angiotensin II and beneficial heptapeptides A(1-7) and Alamandine in the renin-angiotensin system (RAS). In previous studies using in vivo rabbit-iliac arterial models, we showed that Na+ or K+ salts of selected Bisartans initiate a potent dose-response inhibition of vasoconstriction. Furthermore, computational studies revealed that bisartans undergo stable binding to the vital interfacial region between ACE2 and the SARS-CoV-2 "receptor binding domain" (i.e., the viral RBD). Thus, bisartan homologs are expected to interfere with SARS-CoV-2 infection and/or suppress disease expression in humans. The primary goal of this study was to investigate the role of tetrazole in binding and the network of amino acids of SARS-CoV-2 Spike RBD-ACE2 complex involved in interactions with sartans. This study would, furthermore, allow the expansion of the synthetic space to create a diverse suite of new bisartans in conjunction with detailed computational and in vitro antiviral studies. A critical role for tetrazole was uncovered in this study, shedding light on the vital importance of this group in the binding of sartans and bisartans to the ACE2/Spike complex. The in silico data predicting an interaction of tetrazole-containing sartans with ACE2 were experimentally validated by the results of surface plasmon resonance (SPR) analyses performed with a recombinant human ACE2 protein.

Actions of Novel Angiotensin Receptor Blocking Drugs, Bisartans, Relevant for COVID-19 Therapy: Biased Agonism at Angiotensin Receptors and the Beneficial Effects of Neprilysin in the Renin Angiotensin System.

Angiotensin receptor blockers (ARBs) used in the treatment of hypertension and potentially in SARS-CoV-2 infection exhibit inverse agonist effects at angiotensin AR1 receptors, suggesting the receptor may have evolved to accommodate naturally occurring angiotensin 'antipeptides'. Screening of the human genome has identified a peptide (EGVYVHPV) encoded by mRNA, complementary to that encoding ANG II itself, which is an inverse agonist. Thus, opposite strands of DNA encode peptides with opposite effects at AR1 receptors. Agonism and inverse agonism at AR1 receptors can be explained by a receptor 'switching' between an activated state invoking receptor dimerization/G protein coupling and an inverse agonist state mediated by an alternative/second messenger that is slow to reverse. Both receptor states appear to be driven by the formation of the ANG II charge-relay system involving TyrOH-His/imidazole-Carboxylate (analogous to serine proteases). In this system, tyrosinate species formed are essential for activating AT1 and AT2 receptors. ANGII is also known to bind to the zinc-coordinated metalloprotease angiotensin converting enzyme 2 (ACE2) used by the COVID-19 virus to enter cells. Here we report in silico results demonstrating the binding of a new class of anionic biphenyl-tetrazole sartans ('Bisartans') to the active site zinc atom of the endopeptidase Neprilysin (NEP) involved in regulating hypertension, by modulating humoral levels of beneficial vasoactive peptides in the RAS such as vasodilator angiotensin (1-7). In vivo and modeling evidence further suggest Bisartans can inhibit ANG II-induced pulmonary edema and may be useful in combatting SARS-CoV-2 infection by inhibiting ACE2-mediated viral entry to cells.

Diminazene Aceturate Reduces Angiotensin II Constriction and Interacts with the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Diminazene aceturate (DIZE) is a putative angiotensin-converting enzyme 2 (ACE2) activator and angiotensin type 1 receptor antagonist (AT1R). Its simple chemical structure possesses a negatively charged triazene segment that is homologous to the tetrazole of angiotensin receptor blockers (ARB), which explains its AT1R antagonistic activity. Additionally, the activation of ACE2 by DIZE converts the toxic octapeptide angiotensin II (AngII) to the heptapeptides angiotensin 1-7 and alamandine, which promote vasodilation and maintains homeostatic balance. Due to DIZE's protective cardiovascular and pulmonary effects and its ability to target ACE2 (the predominant receptor utilized by severe acute respiratory syndrome coronavirus 2 to enter host cells), it is a promising treatment for coronavirus 2019 (COVID-19). To determine DIZE's ability to inhibit AngII constriction, in vitro isometric tension analysis was conducted on rabbit iliac arteries incubated with DIZE or candesartan and constricted with cumulative doses of AngII. In silico docking and ligand interaction studies were performed to investigate potential interactions between DIZE and other ARBs with AT1R and the spike protein/ACE2 complex. DIZE, similar to the other ARBs investigated, was able to abolish vasoconstriction in response to AngII and exhibited a binding affinity for the spike protein/ACE2 complex (PDB 6LZ6). These results support the potential of DIZE as a treatment for COVID-19.

Understanding the Driving Forces That Trigger Mutations in SARS-CoV-2: Mutational Energetics and the Role of Arginine Blockers in COVID-19 Therapy.

SARS-CoV-2 is a global challenge due to its ability to mutate into variants that spread more rapidly than the wild-type virus. Because the molecular biology of this virus has been studied in such great detail, it represents an archetypal paradigm for research into new antiviral drug therapies. The rapid evolution of SARS-CoV-2 in the human population is driven, in part, by mutations in the receptor-binding domain (RBD) of the spike (S-) protein, some of which enable tighter binding to angiotensin-converting enzyme (ACE2). More stable RBD-ACE2 association is coupled with accelerated hydrolysis of furin and 3CLpro cleavage sites that augment infection. Non-RBD and non-interfacial mutations assist the S-protein in adopting thermodynamically favorable conformations for stronger binding. The driving forces of key mutations for Alpha, Beta, Gamma, Delta, Kappa, Lambda and Omicron variants, which stabilize the RBD-ACE2 complex, are investigated by free-energy computational approaches, as well as equilibrium and steered molecular dynamic simulations. Considered also are the structural hydropathy traits of the residues in the interface between SARS-CoV-2 RBD and ACE2 protein. Salt bridges and π-π interactions are critical forces that create stronger complexes between the RBD and ACE2. The trend of mutations is the replacement of non-polar hydrophobic interactions with polar hydrophilic interactions, which enhance binding of RBD with ACE2. However, this is not always the case, as conformational landscapes also contribute to a stronger binding. Arginine, the most polar and hydrophilic among the natural amino acids, is the most aggressive mutant amino acid for stronger binding. Arginine blockers, such as traditional sartans that bear anionic tetrazoles and carboxylates, may be ideal candidate drugs for retarding viral infection by weakening S-protein RBD binding to ACE2 and discouraging hydrolysis of cleavage sites. Based on our computational results it is suggested that a new generation of "supersartans", called "bisartans", bearing two anionic biphenyl-tetrazole pharmacophores, are superior to carboxylates in terms of their interactions with viral targets, suggesting their potential as drugs in the treatment of COVID-19. In Brief: This in silico study reviews our understanding of molecular driving forces that trigger mutations in the SARS-CoV-2 virus. It also reports further studies on a new class of "supersartans" referred to herein as "bisartans", bearing two anionic biphenyltetrazole moieties that show potential in models for blocking critical amino acids of mutants, such as arginine, in the Delta variant. Bisartans may also act at other targets essential for viral infection and replication (i.e., ACE2, furin cleavage site and 3CLpro), rendering them potential new drugs for additional experimentation and translation to human clinical trials.

Discovery of a new generation of angiotensin receptor blocking drugs: Receptor mechanisms and in silico binding to enzymes relevant to SARS-CoV-2.

The discovery and facile synthesis of a new class of sartan-like arterial antihypertensive drugs (angiotensin receptor blockers [ARBs]), subsequently referred to as "bisartans" is reported. In vivo results and complementary molecular modelling presented in this communication indicate bisartans may be beneficial for the treatment of not only heart disease, diabetes, renal dysfunction, and related illnesses, but possibly COVID-19. Bisartans are novel bis-alkylated imidazole sartan derivatives bearing dual symmetric anionic biphenyl tetrazole moieties. In silico docking and molecular dynamics studies revealed bisartans exhibited higher binding affinities for the ACE2/spike protein complex (PDB 6LZG) compared to all other known sartans. They also underwent stable docking to the Zn2 + domain of the ACE2 catalytic site as well as the critical interfacial region between ACE2 and the SARS-CoV-2 receptor binding domain. Additionally, semi-stable docking of bisartans at the arginine-rich furin-cleavage site of the SARS-CoV-2 spike protein (residues 681-686) required for virus entry into host cells, suggest bisartans may inhibit furin action thereby retarding viral entry into host cells. Bisartan tetrazole groups surpass nitrile, the pharmacophoric "warhead" of PF-07321332, in its ability to disrupt the cysteine charge relay system of 3CLpro. However, despite the apparent targeting of multifunctional sites, bisartans do not inhibit SARS-CoV-2 infection in bioassays as effectively as PF-07321332 (Paxlovid).

Novel Approaches in the Immunotherapy of Multiple Sclerosis: Cyclization of Myelin Epitope Peptides and Conjugation with Mannan.

Multiple Sclerosis (MS) is a serious autoimmune disease. The patient in an advanced state of the disease has restrained mobility and remains handicapped. It is therefore understandable that there is a great need for novel drugs and vaccines for the treatment of MS. Herein we summarise two major approaches applied for the treatment of the disease using peptide molecules alone or conjugated with mannan. The first approach focuses on selective myelin epitope peptide or peptide mimetic therapy alone or conjugated with mannan, and the second on immune-therapy by preventing or controlling disease through the release of appropriate cytokines. In both approaches the use of cyclic peptides offers the advantage of increased stability from proteolytic enzymes. In these approaches, the synthesis of myelin epitope peptides conjugated to mannan is of particular interest as this was found to protect mice against experimental autoimmune encephalomyelitis, an animal model of MS, in prophylactic and therapeutic protocols. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation. The aim of the studies of these peptide epitope analogs is to understand their molecular basis of interactions with human autoimmune T-cell receptor and a MS-associated human leucocyte antigen (HLA)-DR2b. This knowledge will lead the rational design to new beneficial non-peptide mimetic analogs for the treatment of MS. Some issues of the use of nanotechnology will also be addressed as a future trend to tackle the disease. We highlight novel immunomodulation and vaccine-based research against MS based on myelin epitope peptides and strategies developed in our laboratories.