Structural basis of IL-23 antagonism by an Alphabody protein scaffold

Abstract

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein–protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

Cytokines in psoriasis

“…One of the key functions of IL-23 appears to be its ability to amplify and sustain Th17 T cells [97] and as such, is targeted by the anti-IL12p40 biologic ustekinumab (Fig. 3) as well as the more specific approach of targeting the IL-23p19 subunit (CNTO1959 currently in phase 2 trials for palmar plantar pustulosis) and the IL-23 receptor itself [99,100] as a means of targeting IL-17 activity upstream of Th17 cells.…”

Designing helical peptide inhibitors of protein–protein interactions

“…One strategy is to incorporate a helical motif into a larger folded protein scaffold, which can also introduce additional favorable inhibitor-target contacts. [9,10] To promote cell entry, however, it is preferable to minimize peptide molecular weight. For this reason, many methods have been developed to stabilize short peptides in a helical conformation.…”

Polyproteins in structural biology

“…In this study, the efflux pump was assembled by preparing two single-chain polypeptide fusions, AcrB-AcrA-AcrB and 142 New constructs and expressions of proteins Linking individual proteins into polypeptide chains. Proteins engineered into single polypeptide chains were used to obtain suitable sample for structure determination of AcrABZ complex [8 ], an alphabody (MA12) to neutralize human interleukin Il-23 (p19 and p40) [13] and the first structure of a histone-modifying enzyme, the Polycomb Repressive Complex (PRC) 1 ubiquitylation module, bound to a nucleosome [12 ]. Linker amino acid segments are marked (L, L1, L2).…”

“…Polyprotein fusions which are not processed by protease, but remain covalently conjoined by engineered linkers have been instrumental to obtain important insight into numerous essential physiological processes including multidrug efflux, co-translational protein targeting or enzymatic processing of chromatin, among others [8 ,9,10,11 ,12 ]. Moreover, single-chain engineering approaches linking protein domains into novel, artificial polyproteins have resulted in new classes of high-affinity binder molecules as potential protein therapeutics [13] and accelerated elucidation of mechanisms governing protein folding by single-molecule techniques [14]. Thus, polyprotein technologies have recently gained prominence as particularly useful tools for unlocking previously often inaccessible protein samples to detailed structural and mechanistic studies, as illustrated in the following.…”