ABSTRACT
New pathogens responsible for novel human disease outbreaks in the last two decades are mainly the respiratory system viruses. Not different was the last pandemic episode, caused by infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One of the extensively explored targets, in the recent scientific literature, as a possible way for rapid development of COVID-19 specific drug(s) is the interaction between the receptor-binding domain of the virus’ spike (S) glycoprotein and human receptor angiotensin-converting enzyme 2 (hACE2). This protein-protein recognition process is involved in the early stages of the SARS-CoV-2 life cycle leading to the host cell membrane penetration. Thus, disrupting this interaction may block or significantly reduce the infection caused by the novel pathogen. Previously we have designed (by in silico structure-based analysis) three very short peptides having sequences inspirited by hACE2 native fragments, which effectively bind to the SARS-CoV-2 S protein and block its interaction with the human receptor. In continuation of the above mentioned studies, here we presented an application of molecular modeling approach resulting in improved binding affinity of the previously proposed ligand and its enhanced ability to inhibit meaningful host-virus protein-protein interaction. The new optimized hexapeptide binds to the virus protein with affinity one magnitude higher than the initial ligand and, as a very short peptide, has also great potential for further drug development. The peptide-based strategy is rapid and cost-effective for developing and optimizing efficient protein-protein interactions disruptors and may be successfully applied to discover antiviral candidates against other future emerging human viral infections.
ABSTRACT
Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.