Identification of high-quality lead-like molecules depends critically on efficient hit generation and characterization through robust biophysical methods. A suite of biophysical techniques - including NMR, SPR, ITC, TSA, ASMS have been used to profile early hits with different mechanisms of action during the initial phases of hit discovery. However, each technology has its pros and cons that confer significant challenges with respect to biophysical characterization. In this presentation, strategies to overcome challenges with different biophysical platforms are discussed and their application to challenging targets.

My presentation surfaces the weaknesses and blind spots of both small molecule and genetic screening. I offer mitigation strategies, when available, to address existing limitations and propose a framework to decide upon how best to apply each approach.

GPCRs present unique challenges to drug discovery due to their low expression, complexity, and poor stability. Detergent extraction from their native environment is commonplace but can prohibit basic biochemical characterization and render false positives in downstream screening campaigns. To address this problem, we have implemented membrane mimics to generate detergent free GPCRs to facilitate their characterization, enable Hit ID screening campaigns and validation follow-up.

Validating molecular interactions remains a central challenge in drug discovery, especially for targets with elusive or transient binding profiles. This talk explores biophysical strategies to confirm hits, drawing from recent work on on-DNA binder confirmation and mechanistic triage. Drawing from recent case studies and mechanistic insights, we explore how tailored assays and orthogonal approaches can increase confidence in hit follow-up and accelerate lead optimization.

A case study shown here is screening with ASC protein, a critical adapter protein in inflammasome activation, making it a promising target for autoimmune disorders. With its PYD and CARD domains providing potential PPI interfaces for drug design, our strategy targets ASC inhibition by disrupting poly-filament formation through covalent binding at Cys173 on its CARD domain. The comprehensive efforts from primary screening, hit validation to fragment-to-lead expansion will be presented.

Covalent drug discovery benefits from early access to quantitative kinetic information, yet existing approaches often limit throughput and complicate interpretation. New methodologies integrating fragment-based discovery with kinetic analysis are described. A mass spectrometry–based diagonal dose–response time-course (dDRTC) enables practical measurement of kinact/KI for covalent fragments and leads. Surface plasmon resonance (SPR) supports later-stage discovery by providing high-resolution characterization of covalent engagement to guide lead optimization.

By removing the synthesis bottleneck associated with traditional hit-to-lead optimization, high-throughput synthesis and direct-to-biology (D2B) screening accelerates the discovery of drug candidates with first-in-class structures and mechanisms. Building on the success of D2B implemented at Carmot Therapeutics, we present Kimia’s ATLAS platform, a next-generation D2B and direct-to-ADME (D2A) platform that integrates multiple chemistries and machine learning to guide the search for novel drugs, exemplifying the approach with several case studies.

Molecular glues modulate protein proximity, yet have resisted function-first screening.  Here, we present a direct-to-biology approach which can distinguish glues from non-glues. Using high-throughput synthesis and affinity-selection mass spectrometry, we can identify a molecular glue from within 20,000 reaction mixtures. Orthogonal assays confirm gluing behavior. Our results outline a roadmap for de novo glue discovery via kinetic profiling of unpurified small molecules against protein pairs.