Exponential Combination of a and e/g 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.


Peptide permeability assay:
The PAMPA assay measures permeability across an artificial membrane.This method provides an in vitro model for passive diffusion.Passive diffusion is an important factor in determining transport through the gastrointestinal tract, penetration of the blood brain barrier, as well as transport across cell membranes.Permeability can also be influenced by several other mechanisms including paracellular transport and active uptake or efflux which are not assessed in PAMPA.Therefore, PAMPA provides a simplistic approach to permeability by only measuring a single mechanism.This avoids the complexities of active transport/efflux and enables the compounds to be ranked on a single permeability property.As the experimental data evidenced in Table S1, ATF3W-aeg peptide exhibited a less efficient permeability of 1.4nm/s and less membrane retention since mass retentions was 5.8% compared to caffeine as control.However, the ATF3W-aeg peptide inhibitor still can be categorised into high permeability as Log(Permeability) of ATF3W-aeg at -5.9 which was larger than -6 according to the industry standard recommended in the manual of the BD™ pre-coated PAMPA plate system.TableS1: Shown are peptide permeability assay on ATF3W-aeg.Permeability showed the permeable speed of peptide through membrane and mass retention showed percentage of peptide holding on the membrane.
Peptide permeability assay: To evaluate the peptide permeability, the BD™ pre-coated PAMPA plate system was employed.The 300uL of 200uM peptide in PBS buffer were added in the well of the receiver plate, and slowly placed the filter plate with the 200uL PBS buffer per well on the receiver plate.The peptide concentrations in both plates were determined by UV spectroscopy after the assembly plate system were incubated at room temperature for 5 hours, and the peptide permeability and mass retention were calculated by using the PAMPA plate system formula which recommended in the manufacture manual.
Permeability (in unit of cm/s): -ln  Serum stability: Peptide stocks (600 uM) were prepared in water, and 75 uL was added to 1425 uL human serum (Merck) before incubation at 37 °C.100 uL of aliquots were removed at designated time-points and added to 300 uL of acetonitrile: water (3:1) and centrifuged (18,000 x g, 15 min).
The supernatant was analysed by LC-MS, and the peptide was quantified as the sum of the peaks with the two largest intensities 1 .

Fig S1 :
Fig S1: Constructed PCA library a and library e/g are shown, along with two sequences verified by DNA sequencing to check accuracy and sequence variation