Structure-activity analysis of truncated albumin-binding domains suggests new lead constructs for potential therapeutic delivery

Conan K Wang,Anna S Amiss,Joachim Weidmann,David J Craik

doi:10.1074/jbc.ra120.014168

Abstract

Rapid clearance by renal filtration is a major impediment to the translation of small bioactive biologics into drugs. To extend serum t1/2, a commonly used approach is to attach drug leads to the G-related albumin-binding domain (ABD) to bind albumin and evade clearance. Despite the success of this approach in extending half-lives of a wide range of biologics, it is unclear whether the existing constructs are optimized for binding and size; any improvements along these lines could lead to improved drugs. Characterization of the biophysics of binding of an ABD to albumin in solution could shed light on this question. Here, we examine the binding of an ABD to human serum albumin using isothermal titration calorimetry and assess the structural integrity of the ABD using CD, NMR, and molecular dynamics. A structure-activity analysis of truncations of the ABD suggests that downsized variants could replace the full-length domain. Reducing size could have the benefit of reducing potential immunogenicity problems. We further showed that one of these variants could be used to design a bifunctional molecule with affinity for albumin and a serum protein involved in cholesterol metabolism, PCSK9, demonstrating the potential utility of these fragments in the design of cholesterol-lowering drugs. Future work could extend these in vitro binding studies to other ABD variants to develop therapeutics. Our study presents new understanding of the solution structural and binding properties of ABDs, which has implications for the design of next-generation long-lasting therapeutics.

Highlights

Peptides and small proteins have great potential as therapeutic leads because of their high target specificities [1, 2]
ABD23 appeared to be the most promising albumin-binding domain (ABD) truncation to be constrained, because the crystal structure pinpoints it to be the minimal region of ABD required for binding to human serum albumin (Fig. 1)
ABD23 is the most likely to bind to the same interface on albumin as the full-length domain indicated by the crystal structure, because it incorporates all residues at the contact interface (Fig. 1a)

Summary

Introduction

Peptides and small proteins have great potential as therapeutic leads because of their high target specificities [1, 2]. We obtained thermodynamic parameters (e.g. enthalpy change, DH, and entropy change, DS) as well as the dissociation constant (Kd) and stoichiometry (N) of binding for each synthesized peptide to human serum albumin (Fig. 3 and Fig. S2). Titration of the full-length domain (ABD) into albumin showed that binding was enthalpy driven (DH = 250.4 6 3.0 kcal/mol, DS = 2149 6 10 cal/mol/K; Fig. 3a and Table 1).

Results

Conclusion