A Rationally Designed Synthetic Antiviral Peptide Binder Targeting the Receptor-Binding Domain of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel coronavirus, is the causative agent responsible for the spread of the COVID19 pandemic across the globe. The global impact of the COVID19 pandemic, the successful approval of vaccines for controlling the pandemic, and the further resurgence of COVID19 necessitate the exploration and validation of alternative therapeutic avenues targeting SARS-CoV-2. The initial entry and further invasion by SARS-CoV-2 require strong protein-protein interactions (PPIs) between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptors expressed on the cell surfaces of various tissues. In principle, disruption of the PPIs between the RBD of SARS-CoV-2 and the ACE2 receptor by designer peptides with optimized pharmacology appears to be an ideal choice for potentially preventing viral entry with minimal immunogenicity. In this context, the current study describes a short, synthetic designer peptide (codenamed SR16, ≤18 aa, molecular weight ≤2.5 kDa), which has a few noncoded amino acids, demonstrates a helical conformation in solution, and also engages the RBD of SARS-CoV-2 through a high-affinity interaction, as judged from a battery of biophysical studies. Further, the designer peptide demonstrates resistance to trypsin degradation, appears to be nontoxic to mammalian cells, and also does not induce hemolysis in freshly isolated human erythrocytes. In summary, SR16 appears to be an ideal peptide binder targeting the RBD of SARS-CoV-2, which has the potential for further optimization and development as an antiviral agent targeting SARS-CoV-2.

Rational design of antibody-like peptides for targeting the human complement fragment protein C5a.

Human complement fragment 5a (C5a) is one of the most potent glycoproteins generated downstream of C3a and C4a during late-stage activation of the complement signaling cascade. C5a recruits receptors like C5aR1 and C5aR2 and is established to play a critical role in complement-mediated inflammation. Thus, excessive C5a in the plasma due to aberrant activation of the complement contributes to the pathophysiology of several chronic inflammatory diseases. Therefore, restricting the excessive interaction of C5a with its receptors by neutralizing C5a has been one of the most effective therapeutic strategies for the management of inflammatory diseases. Indeed, antibodies targeting C5 (Eculizumab), the precursor of C5a, and C5a (Vilobelimab) have already been approved by the FDA. Still, small designer peptides that work like antibodies and can target and stop C5a from interacting with its receptors seem to be a possible therapeutic alternative to antibodies because they are smaller, cheaper to make, more specific to their target, and can get through membrane barriers. As a proof-of-principle, the current study describes the computational design and evaluation of a pair of peptides that are able to form stable high-affinity complexes with the epitope regions of C5a that are important for the recruitment of C5aR1 and C5aR2. The computational data further supports the potential of designer peptides for mimicking the function of antibodies targeting C5a. However, further experimental studies will be required to establish the structure-function relationship of the designer peptides and also to establish the hypothesis of antibody-like peptides targeting C5a.

A rationally engineered small antimicrobial peptide with potent antibacterial activity.

Antimicrobial resistance (AMR) is a silent pandemic declared by the WHO that requires urgent attention in the post-COVID world. AMR is a critical public health concern worldwide, potentially affecting people at different stages of life, including the veterinary and agriculture industries. Notably, very few new-age antimicrobial agents are in the current developmental pipeline. Thus, the design, discovery, and development of new antimicrobial agents are required to address the menace of AMR. Antimicrobial peptides (AMPs) are an important class of antimicrobial agents for combating AMR due to their broad-spectrum activity and ability to evade AMR through a multimodal mechanism of action. However, molecular size, aggregability, proteolytic degradation, cytotoxicity, and hemolysis activity significantly limit the clinical application of natural AMPs. The de novo design and engineering of a short synthetic amphipathic AMP (≤16 aa, Mol. Wt. ≤ 2 kDa) with an unusual architecture comprised of coded and noncoded amino acids (NCAAs) is presented here, which demonstrates potent antibacterial activity against a few selected bacterial strains mentioned in the WHO priority list. The designer AMP is conformationally ordered in solution and effectively permeabilizes the outer and inner membranes, leading to bacterial growth inhibition and death. Additionally, the peptide is resistant to proteolysis and has negligible cytotoxicity and hemolysis activity up to 150 µM toward cultured human cell lines and erythrocytes. The designer AMP is unique and appears to be a potent therapeutic candidate, which can be subsequently subjected to preclinical studies to explicitly understand and address the menace of AMR.

The anaphylatoxin C5a: Structure, function, signaling, physiology, disease, and therapeutics.

The complement system is one of the oldest known tightly regulated host defense systems evolved for efficiently functioning cell-based immune systems and antibodies. Essentially, the complement system acts as a pivot between the innate and adaptive arms of the immune system. The complement system collectively represents a cocktail of ∼50 cell-bound/soluble glycoproteins directly involved in controlling infection and inflammation. Activation of the complement cascade generates complement fragments like C3a, C4a, and C5a as anaphylatoxins. C5a is the most potent proinflammatory anaphylatoxin, which is involved in inflammatory signaling in a myriad of tissues. This review provides a comprehensive overview of human C5a in the context of its structure and signaling under several pathophysiological conditions, including the current and future therapeutic applications targeting C5a.

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2.

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or ß-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a-C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.

Deciphering the conformational landscape of few selected aromatic noncoded amino acids (NCAAs) for applications in rational design of peptide therapeutics.

Amino acids are the essential building blocks of both synthetic and natural peptides, which are crucial for biological functions and also important as biological probes for mapping the complex protein-protein interactions (PPIs) in both prokaryotic and eukaryotic systems. Mapping the PPIs through the chemical biology approach provides pharmacologically relevant peptides, which can have agonistic or antagonistic effects on the targeted biological systems. It is evidenced that ≥ 60 peptide-based drugs have been approved by the US-FDA so far, and the number will improve further in the foreseeable future, as ≥ 140 peptides are currently in clinical trials. However, natural peptides often require fine-tuning of their pharmacological properties by strategically replacing the αL-amino acids of the peptides with non-coded amino acids (NCAA), for which codons are absent in the genetic code for biosynthesis of proteins, prior to their applications as therapeutics. Considering the diverse repertoire of the NCAAs, the conformational space of many NCAAs is yet to be explored systematically in the context of the rational design of therapeutic peptides. The current study deciphers the conformational landscape of a few such Cα-substituted aromatic NCAAs (Ing: 2-indanyl-L-Glycine; Bpa: 4-benzoyl-L-phenylalanine; Aic: 2-aminoindane-2-carboxylic acid) both in the context of tripeptides and model synthetic peptide sequences, using alanine (Ala) and proline (Pro) as the reference. The combined data obtained from the computational and biophysical studies indicate the general success of this approach, which can be exploited further to rationally design optimized peptide sequences of unusual architecture with potent antimicrobial, antiviral, gluco-regulatory, immunomodulatory, and anti-inflammatory activities.

Low clarithromycin resistance in virulent Helicobacter pylori from dyspeptic patients at a tertiary care centre in Odisha.

PURPOSE: Universal eradication or use of failing antibiotic can add fuel to the antimicrobial resistance pandemic. Outcome of Helicobacter pylori (HP) infection depends at least partly virulence factors and its eradication as preventive measure against gastric cancer is advocated by some guidelines. There is need to identify candidates at risk for gastric cancer and antimicrobial resistance in HP for rational management. Such candidates could be identified by studying the association of virulence factors with clinical outcome. As this data is lacking from Odisha this study was undertaken. METHODS: 113 consecutive dyspeptic patients who underwent endoscopy at our hospital were recruited to obtain gastric biopsies for culture and antibiotic susceptibility, histological examination, molecular detection of HP, virulence typing (cagA, EPIYA typing, vacA, vacAs1/s2, vacAm1/m2 and babA2) by conventional PCR and identification of clarithromycin resistance by real-time PCR. Cultured isolates were subjected to antibiotic sensitivity using e strips as per EUCAST guidelines. RESULTS: 93 (82.3%) dyspeptic patients were infected by HP by histology & PCR, while 90 (79.6%) were rapid urea test positive, and HP was cultured from 32 (28.3%) of these patients. Eleven (11.8%) of the 93 samples with HP were resistant to clarithromycin by real-time PCR. Of the 93 patients with HP infection by histopathology and PCR, 62 (66.7%), 87(93.5%) and 43 (46.2%) harboured cagA, vacA and babA2 genes. The western cagA found in 33 (35.5%) samples and vacAs1m1 in 50 (53.8%) samples were the commonest virulence subtypes. No association was found between virulence factors and outcome except vacAs2m2 and vac s1/m1m2, which were significantly associated with peptic ulcers. Phenotypically 11(34.4%), 1(3.1%), 21(65.6%) and 26 (81.2%) isolates were resistant to clarithromycin, amoxicillin, levofloxacin, and metronidazole. CONCLUSIONS: This is the first study that explored the antibiotic resistance of HP, and its virulence factors in dyspeptic patients from this region of India.