Binding of raloxifene to human complement fragment 5a (^hC5a): a perspective on cytokine storm and COVID19

Abstract

Human C5a (h C5a), one of the pro-inflammatory glycoproteins of the complement system is known to undergo production hyperdrive in response to stress and infection. h C5a has been associated with the pathogenesis of many chronic and acute diseases, due to its proven ability in triggering the 'cytokine storm', by binding to its cognate receptor C5aR, expressed in myriad of tissues. Given the pleiotropic downstream function of h C5a, it is logical to consider the h C5a or its precursors as potential drug targets, and thus, we have been rationally pursuing the idea of neutralizing the harmful effect of excessive h C5a, by implementing the repurposing strategies for FDA-approved drugs. Indeed, the proof of principle biophysical studies published recently is encouraging, which strongly supports the potential of this strategy. Considering BSA-carprofen as a reference model system, the current study further explores the inherent conformational plasticity of h C5a and its effect in accommodating more than one drug molecule cooperatively at multiple sites. The data generated by recruiting a battery of experimental and computational biology techniques strongly suggest that h C5a can sequentially accommodate more than one raloxifene molecule with an estimated Ki $ 0.5 mM and Ki $ 3.58 mM on its surface at non-analogous sites. The study hints at exploration of polypharmacology approach, as a new avenue for discovering synergistic drug molecule pairs, or drug molecules with 'broad-range' binding affinity for targeting the different 'hot spots' on h C5a, as an alternative combination therapy for possible management of the 'cytokine storm'-related inflammatory diseases, like COVID19.

Interaction of Human C5a with the Major Peptide Fragments of C5aR1: Direct Evidence in Support of “Two-Site” Binding Paradigm

“…Therapeutic intervention of the C5a–C5aR1 system usually involves the following strategies: (i) block the ECS of C5aR1, (ii) block the generation of C5a, and (iii) block or neutralize the C5a. − Large-scale mutagenesis and biomolecular signaling data evidence that the nanomolar to picomolar potency of C5a toward C5aR1 is due to the recruitment of two distal sites with large surface areas, respectively, engaging (i) the NT-peptide (Site1) and (ii) ECS of C5aR1 (Site2) with the core and the C-terminus (CT) peptide of C5a through a specific protein–protein interaction. In addition, extensive mutational studies − on both C5a and C5aR1 had provided the following two key observations: (i) truncation of the NT-peptide region significantly affects the affinity of C5aR1 toward C5a, and the NT-peptide of C5aR1 is very important for activation by C5a, , (ii) the NT-truncated C5aR1 could be effectively activated by short peptide analogues based on the CT-region of C5a albeit with relatively weak binding affinity. , Therefore, it was hypothesized that the NT-peptide contains the first binding site (Site1), and the poorly defined interhelical crevice in the transmembrane region contains the second binding site (Site2) on C5aR1 for recognizing the C5a to trigger the downstream biomolecular signaling.…”

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

“…In fact, Eculizumab (Soliris; Alexion Pharmaceuticals) and IFX-1 (InflaRx) are the two FDA-approved antibodies currently available in the market that respectively targets C5, the precursor protein of C5a, or the C5a directly. C5a being an important protein of the immune system, completely shutting it down through antibodies is physiologically undesired, and thus, we have been actively pursuing the avenue of repurposing the FDA-approved drugs as potential “neutraligands” of C5a (Das and Rana 2021 ; Mishra and Rana 2019 ; Mishra et al 2022 ) for therapeutically modulating the pathological stimulation of C5a-C5aR1 signaling axes. Nevertheless, antisense peptides (Fujita et al 2004 ) targeting the C5a have been explored earlier, and thus, in the context of the PPIs involving C5a and C5aR1 (Das et al 2021 ; Sahoo et al 2018 ; Rana and Sahoo 2015 ), the excessive C5a produced in the body can also be neutralized with designer peptides (Fig.…”

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

“…Although complementary designer peptides targeting the antisense homology box of C5a have shown some exciting results earlier in the cell culture and animal model studies, 30,31 except the anti-C5a antibody (Vilobelimab) 10 nothing is currently known to target C5a directly. In our previous studies, we have shown that few selective drug molecules, such as raloxifene (Ral: K d ~0.71 μM) 17,19 and prednisone (PDN: K d ~0.38 μM), 18 can act as neutraligands by binding and altering the conformation of C5a, which can potentially modulate the interaction of C5a with the complement receptors C5aR1 and C5aR2. Thus, in the current study, we wanted to explore whether the binding of neutraligands to C5a will affect the interaction of the peptides derived from the complement receptors, as hypothesized earlier for the C5aR1 17 and schematically illustrated in Figure 11 for C5aR2.…”

“…In addition, we had earlier demonstrated that selected small-molecule drugs such as raloxifene and prednisone could act as neutraligands by binding and inducing conformational changes in C5a, which can affect the interaction of C5a with its receptors. [17][18][19] Unlike antibodies, targeting C5a with small molecule drugs for modulating the pathophysiological axes of C5a-C5aR1 or C5a-C5aR2 for therapeutic benefit is a relatively new concept that needs further exploration. Thus, the current study further explores the idea of documenting whether the presence of neutraligands can modulate the interaction of C5a with the respective peptide fragments of C5aR1 and C5aR2.…”