1 research outputs found
Exponential Combination of <i>a</i> and <i>e/g</i> Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor
Activating transcription factor 3 (ATF3) is an activation
transcription
factor/cyclic adenosine monophosphate (cAMP) responsive element-binding
(CREB) protein family member. It is recognized as an important regulator
of cancer progression by repressing expression of key inflammatory
factors such as interferon-γ and chemokine (C–C motif)
ligand 4 (CCL4). Here, we describe a novel library screening approach
that probes individual leucine zipper components before combining
them to search exponentially larger sequence spaces not normally accessible
to intracellular screening. To do so, we employ two individual semirational
library design approaches and screen using a protein-fragment complementation
assay (PCA). First, a 248,832-member library explored 12 amino acid
positions at all five a positions to identify those
that provided improved binding, with all e/g positions
fixed as Q, placing selection pressure onto the library options provided.
Next, a 59,049-member library probed all ten e/g positions
with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence
pattern (AIAIA), weakly favoring leucine zipper formation, while placing
selection pressure onto e/g options provided. The
combined a/e/g library represents ∼14.7 billion
members, with the resulting peptide, ATF3W_aeg, binding ATF3 with
high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization.
Moreover, ATF3W_aeg is notably improved over component PCA hits, with
target specificity found to be driven predominantly by electrostatic
interactions. The combined a/e/g exponential library
screening approach provides a robust, accelerated platform for exploring
larger peptide libraries, toward derivation of potent yet selective
antagonists that avoid homoassociation to provide new insight into
rational peptide design