Structure of Designer Antibody-like Peptides Binding to the Human C5a with Potential to Modulate the C5a Receptor Signaling.

C5a is an integral glycoprotein of the complement system that plays an important role in inflammation and immunity. The physiological concentration of C5a is observed to be elevated under various immunoinflammatory pathophysiological conditions in humans. The pathophysiology of C5a is linked to the "two-site" protein-protein interactions (PPIs) with two genomically related receptors, such as C5aR1 and C5aR2. Therefore, pharmacophores that can potentially block the PPIs between C5a-C5aR1 and C5a-C5aR2 have tremendous potential for development as future therapeutics. Notably, the FDA has already approved antibodies that target the precursors of C5a (Eculizumab, 148 kDa) and C5a (Vilobelimab, 149 kDa) for marketing as complement-targeted therapeutics. In this context, the current study reports the structural characterization of a pair of synthetic designer antibody-like peptides (DePA and DePA1; ≤3.8 kDa) that bind to hotspot regions on C5a and also demonstrates potential traits to neutralize the function of C5a under pathophysiological conditions.

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.

Conformational variants of the ternary complex of C5a, C5aR1, and G-protein.

The complement component fragment 5a (C5a) binds and activates two complement receptors like C5aR1 and C5aR2, which play a significant role in orchestrating the proinflammatory function of C5a in tissues through the recruitment of heterotrimeric G-proteins and ß-arrestins. Dysregulation of the complement induces excessive production of C5a, which triggers aberrant activation of the C5a-C5aR1-G-protein and C5a-C5aR2-ß-arrestin signalling axes in tissues, contributing to the pathology of numerous immune-inflammatory diseases. Thus, understanding the interaction of C5a with C5aR1 and C5aR2, as well as the interaction of G-protein and ß-arrestins, respectively, with C5a-C5aR1 and C5a-C5aR2, holds tremendous therapeutic value. In the absence of structural data, we have previously elaborated the binary complexes of C5a-C5aR1 and C5a-C5aR2, as well as the ternary complex of C5a-C5aR2-ß-arrestin1, in highly refined model structures. While our ternary model complex of C5a-C5aR1-G-protein was in progress, two cryo-electron microscopy-based ternary structural complexes of C5aR1 were made available by others. However, it is observed that the interaction of the crucial NT-peptide of C5aR1 with C5a, including the portion of the G⍺i-subunit that harbors the switch-I region, is not fully resolved in both complexes. The current study addresses the issues and provides two highly refined alternative model ternary complexes of C5a-C5aR1-G-protein. The study highlights the conformational heterogeneity in C5aR1 by comparing the two conformational variants of the model ternary complex in the context of C5a-C5aR2-ß-arrestin1 for further devising methods and molecules targeting both surface and intracellular C5aR1/C5aR2 for effectively mitigating the proinflammatory role of C5a in various disease settings.Communicated by Ramaswamy H. Sarma.

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.

Ternary model structural complex of C5a, C5aR2, and ß-arrestin1.

Complement component fragment 5a (C5a) is one of the potent proinflammatory modulators of the complement system. C5a recruits two genomically related G protein-coupled receptors (GPCRs), like C5aR1 and C5aR2, constituting a binary complex. The C5a-C5aR1/C5aR2 binary complexes involve other transducer proteins like heterotrimeric G-proteins and ß-arrestins to generate the fully active ternary complexes that trigger intracellular signaling through downstream effector molecules in tissues. In the absence of structural data, we had recently developed highly refined model structures of C5aR2 in its inactive (free), meta-active (complexed to the CT-peptide of C5a), and active (complexed to C5a) state embedded to a model palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. Compared to C5aR1, C5aR2 is established as a noncanonical GPCR, as it recruits and signals through ß-arrestins rather than G-proteins. Notably, structural understanding of the ternary complex involving C5a-C5aR2-ß-arrestin is currently unknown. The current study has attempted to fill the gap by generating a highly refined, fully active ternary model structural complex of the C5a-C5aR2-ß-arrestin1 embedded in a model POPC bilayer. The computational modeling, 500 ns molecular dynamics (MD) studies, and the principal component analysis (PCA), including the molecular mechanics Poisson-Boltzmann surface area (MM PBSA) based data presented in this study, provide an experimentally testable hypothesis about C5a-C5aR2-ß-arrestin1 extendable to other such ternary systems. The model ternary complex of C5a-C5aR2-ß-arrestin1 will further enrich the current structural understanding related to the interaction of ß-arrestins with the C5a-C5aR2 system.Communicated by Ramaswamy H. Sarma.

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.