Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function

Abstract

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.

Deriving Immune Modulating Drugs from Viruses—A New Class of Biologics

“…Serpins function as suicide inhibitors providing an external strained reactive center loop (RCL) that presents a P1-P1 scissile bond as bait for cleavage by targeted proteases [31]. The proteases that are targeted by serpins then cleave the RCL at the P1-P1 bond, becoming covalently bound to the cleaved end of the RCL in a Michaelis complex, which is normally transient in an enzyme-substrate complex [33,34]. The cleaved RCL with the bound protease then folds in a dramatic rearrangement into the A β-sheet (as the third of five strands), bringing the now-deformed and neutralized protease a distance of more than 70Å to the opposite pole of the serpin in a stable, inactive serpin-enzyme complex [35] (Figure 3).…”

“…The cleaved RCL with the bound protease then folds in a dramatic rearrangement into the A β-sheet (as the third of five strands), bringing the now-deformed and neutralized protease a distance of more than 70Å to the opposite pole of the serpin in a stable, inactive serpin-enzyme complex [35] (Figure 3). in an enzyme-substrate complex [33,34]. The cleaved RCL with the bound protease then folds in a dramatic rearrangement into the A β-sheet (as the third of five strands), bringing the nowdeformed and neutralized protease a distance of more than 70Å to the opposite pole of the serpin in a stable, inactive serpin-enzyme complex [35] (Figure 3).…”

“…Thus, 1 to 10 days of Serp-1 treatment given systemically has had up to 30 days benefit, and for the inflammatory vasculitic syndrome (IVS) model of herpesvirus-induced vasculitis, 10 to 30 days of Serp-1 treatment led to improved survival up to 150 days [56]. A series of Serp-1 and mammalian neuroserpin (NSP) RCL peptides were thus tested; peptide sequences based upon natural cleavage sites predicted by the Expasy/PeptideCutter program [33,66]. One of the Serp-1 RCL peptides, designated S-7, reduced inflammation after aortic allograft transplant in mice, as well as improved survival with similar efficacy to Serp-1 in the MHV-68 IVS mouse model in IFNγR −/− mice.…”

“…Serp-1 and the Serp-1 S-7 peptide lose therapeutic efficacy and no longer improve survival when the gut bacterial microbiome is suppressed with broad-spectrum antibiotics [33]. This finding suggests that this Myxomaviral immune regulating serpin has tapped into trans-kingdom interactions and host defenses that modify host immune responses between the infecting virus and the host bacterial microbiome.…”

Local Serpin Treatment via Chitosan-Collagen Hydrogel after Spinal Cord Injury Reduces Tissue Damage and Improves Neurologic Function

“…Serine protease inhibitors (serpins) are "suicide" inhibitors that have highly conserved structures and exist in all kingdoms. In mammals, serpins are highly prevalent, represent up to 2%-10% of the proteins in the circulating blood, and function to prevent excess bleeding or clotting [22][23][24][25][26][27][31][32][33]. Inhibition of serine proteases by serpins results in the covalent linkage of the serpin with target proteases which results in a serpin-enzyme complex (SEC) which is then taken up by SEC receptors such as the LDL receptor-related protein 1 expressed by neurons, hepatocytes, macrophages, and other tissues with subsequent metabolic breakdown of the receptor [34,35].…”