Biology subjects

Pixley, J. A.

Publications and source records attributed to Pixley, J. A..

4 recordsLinked to original sources

bioRxiv2026

PANCS-Inhibitors: A rapid method to directly select for protein-protein interaction inhibitors

Aberrant protein-protein interactions (PPIs) drive myriad diseases. Inhibiting these PPIs often relies on discovering molecules that bind to one of the proteins and hoping that this binding inhibits the PPI. Molecular binder discovery often takes months, but a discovery process that ensures that the resulting molecule not only binds a target protein, but selectively inhibits a target PPI, could dramatically accelerate these endeavors. Here, we develop Phage-Assisted Non-Continuous Selection of PPI Inhibitors (PANCS-Inhibitors): a rapid screening platform that directly selects for molecules capable of disrupting a pre-formed PPI. We demonstrate this new platform using three clinically relevant oncogenic PPIs: KRas-Raf, Mdm2-p53, and Myc-Max. PANCS-Inhibitors can be used to both improve known PPI inhibitors and for de novo discovery of mini-protein PPI inhibitors that function in mammalian cells. This platform has the potential to rapidly generate inhibitors for many clinically relevant PPIs, which can be used as starting points for therapeutic development.

synthetic biology↗

bioRxiv2025

PANCS-spec-Binders: A system for rapidly discovering isoform- or epitope-specific binders

Proteins that bind to a target protein of interest, termed "binders," are essential components of biological research reagents and therapeutics. Target proteins present multiple binding surfaces with varying interaction potential. High-potential surfaces, or "hot spots," are experimentally identified as the most probable binding sites in de novo discovery campaigns. However, hot spots and their default binding modes do not always confer the desired specificity. Related proteins or isoforms often share similar hot spots, resulting in promiscuous binding. Interaction with a hot spot may also fail to elicit the intended biological outcome. Consequently, methods that direct de novo binder discovery toward targets with defined specificity are critically needed. We recently developed phage-assisted non-continuous selection of binders (PANCS-Binders), a selection platform with unparalleled speed and sequence-function fidelity that enables routine de novo binder discovery within days. However, because PANCS-Binder selections enrich variants based primarily on affinity, secondary screening is unlikely to identify binders to lesser hot spots because of the high likelihood of convergence. These alternative binding surfaces with weaker inherent interactions may possess desirable specificity profiles. Here, we develop PANCS-spec-Binders, which incorporates simultaneous selection and counterselection to control the specificity of enriched binders. We demonstrate PANCS-spec-Binders in two proof- of-concept applications: (1) discovery of isoform-selective binders that bind HRAS with >100-fold higher affinity than the highly related KRAS isoform, and (2) discovery of epitope-specific binders that either target or avoid the LIR interaction region of LC3B. PANCS-spec-Binders enables rapid identification of binders with defined specificity within days. SIGNIFICANCEAffinity reagents, termed "binders", are essential tools in research and therapeutic development. Binders generally require high specificity either at the selectivity level (binding only the target protein, but not related proteins) or at the epitope level (binding only at a specific surface on the target rather than another). While methods to discover binders in general have progressed, identifying binders with defined specificity features often requires extensive secondary screening and frequently results in failure. Here, we adapt our recently developed binder discovery platform to solve these two selectivity problems. By rapidly screening billions of variants, we can direct specificity between highly related proteins, direct binding to a specific epitope, and, because of the fidelity of our selections, identify binders that specifically avoid a defined epitope.

synthetic biology↗

bioRxiv2025

Mapping the diverse topologies of protein-protein interaction fitness landscapes

De novo binder discovery is unpredictable and inefficient due to a lack of quantitative understanding of protein-protein interaction (PPI) sequence-function landscapes. Here, we use our PANCS-Binder technology to perform >1,300 independent selections of various library sizes and compositions of a randomized small protein to identify binders to a panel of 96 distinct target proteins. For successful selections, we discovered reproducible fitness landscapes that group into a few, target-specific, clusters. Each cluster defines a minimal binding motif whose frequency is inversely proportional to the number of specified amino acids ([~]2-8) and determines selection success, which is quantifiable by the density of binders to the target within a theoretical sequence space. We leverage these data to develop a supervised contrastive learning approach that discriminates binders from non-binders and demonstrates generalization beyond a threshold amount of data. Together, this framework renders PPI landscapes measurable and predictive, accelerating de novo binder discovery and optimization.

synthetic biology↗

bioRxiv2025

High-throughput protein binder discovery by rapid in vivo selection

Proteins that selectively bind to a target of interest are foundational components of research pipelines1,2, diagnostics3, and therapeutics4. Current immunization-based5,6, display-based7-14, and computational approaches15-17,18 for discovering binders are laborious and time-consuming - taking months or more, suffer from high false positives - necessitating extensive secondary screening, and have a high failure rate, especially for disordered proteins and other challenging target classes. Here we establish Phage-Assisted Non-Continuous Selection of Protein Binders (PANCS-binders), an in vivo selection platform that links the life cycle of M13 phage to target protein binding though customized proximity-dependent split RNA polymerase biosensors, allowing for complete and comprehensive high-throughput screening of billion-plus member protein variant libraries with high signal-to-noise. We showcase the utility of PANCS-Binders by screening multiple protein libraries each against a panel of 95 separate therapeutically relevant targets, thereby individually assessing over 1011 protein-protein interaction pairs, completed in two days. These selections yielded large, high-quality datasets and hundreds of novel binders, which we showed can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. PANCS-Binders dramatically accelerates and simplifies the binder discovery process, the democratization of which will help unlock new creative potential in proteome-targeting with engineered binder-based biotechnologies.

synthetic biology↗