Self-Assembled Proteomimetic (SAP) with Antibody-like Binding from Short PNA-Peptide Conjugates

<p><span><span>Affinity proteins </span><span>based on </span><span>a </span><span>three-helix </span><span>bundle</span> <span>(</span><span>affibodies, </span><span>alphabodies</span><span> and computationally </span></span><span><span>de novo</span></span><span> <span>designed</span><span> ones)</span><span> have shown to be a general platform to discover binders with properties reminiscent of </span><span>antibodies</span><span>, combining </span><span>high </span><span>target </span><span>specificity</span><span> with </span><span>affinities reaching well below</span> <span>the </span><span>nanomolar</span><span>.</span> <span>Herein</span><span>,</span><span> we report a new strategy</span><span>, coined self-assembled proteomimetic (SAP)</span><span>,</span><span> to mimic </span><span>such</span><span> three-helix bundle</span><span> architecture with a hybridization-enforced two-helix </span><span>coiled</span> <span>coil</span><span> that is obtained by templated</span> <span>native chemical ligation (</span><span>T-</span><span>NCL) of PNA-peptide conjugates.</span> <span>This SAP </span><span>strategy</span> <span>stands out by</span><span> its</span><span> synthetic accessibility reducing the length on the longest </span><span>synthetic</span><span> peptide to </span><span>less than 30 amino acids, readily attainable by standard SPPS methodologies</span><span>. We show that the </span><span>T</span><span>-NCL dramatically accelerates the </span><span>ligation</span><span>, enabling this chemistry to </span><span>proceed</span> <span>in a combinatorial fashion </span><span>at</span><span> low</span> <span>micromolar</span><span> concentration</span><span>s</span><span>.</span> <span>We </span><span>demonstrate</span> <span>that small </span><span>combinatorial </span><span>libraries of </span><span>SAP</span><span>s</span><span> can be prepared in one operation and used directly in </span><span>affinity selection</span><span>s</span><span> against a target of interest </span><span>with an</span><span> LC-MS </span><span>analysis</span><span> of the fittest binders</span><span>.</span> <span>Moreover, we </span><span>show</span><span> that </span><span>the underlying</span> <span>design</span><span> paradigm</span><span> is functional for</span><span> SAPs based on structurally distinct three-helix peptides </span><span>aimed at</span><span> different </span><span>therapeutic </span><span>targets, namely</span> <span>HER2 </span><span>and</span><span> spike&rsquo;s RBD</span><span>,</span></span> <span><span>reaching picomolar </span><span>affinities</span></span><span><span>. We further </span><span>illustrate </span><span>that the</span> <span>affinity </span><span>of the </span><span>S</span><span>AP</span><span> can be allosterically regulated using a toehold displacement</span><span> of the hybridizing PNAs</span><span> to disrupt the </span><span>coiled coil</span><span> stabilization.</span> <span>Finally, w</span><span>e show that </span><span>an RBD-targeting </span><span>SAP effectively inhibits viral </span><span>entry </span><span>of SARS-CoV-2</span> <span>with an IC</span></span><span><span>50</span></span><span><span> of </span><span>2.8</span> <span>nM</span><span>.</span></span><span>&nbsp;</span></p>