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.

Targeting PPIs that are either unstructured or "flat” is a challenge. We use Gasdermin-D (GSDMD) as a case study to demonstrate that transition metal ions can be used to identify novel sites that can disrupt PPI. Upon cleavage by inflammatory caspases, GSDMD oligomerizes on the cell membrane to form pores, triggering pro-inflammatory cytokine release and pyroptotic cell death. Although structures of its full-length form and the pore are known, it has not been trivial to identify any targetable inhibitory site on this protein. Using biochemical and cellular assays, DLS, nanoDSF and cryoEM, we show that GSDMD is inhibited by Ni(II) coordination complexes, uncovering a novel binding site with key insights gained from site-directed mutagenesis.

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.

G-protein-coupled receptors (GPCRs) represent one-third of all FDA approved therapeutics. Structure-based design of partial agonists represents a challenge for the GPCR field. Using natural products medicinal chemistry, single molecule biophysics, pharmacology, and structural biology we have identified an allosteric subpocket in the orthosteric site of the mu opioid receptor that acts as an efficacy regulator. Findings could pave way for a new class of safer analgesics.

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.

Epigenetic proteins are key drug targets in cancer, with approved therapies for leukemia and lymphoma, and emerging applications in neurodegenerative and metabolic diseases. Sirtuin 6 is a chromatin-associated deacetylase that binds DNA in compact chromatin and regulates gene expression. DNA-encoded libraries were screened in living cells under physiological conditions, enabling discovery of selective Sirtuin 6 small-molecule hits and demonstrating the utility of DELs in Cells for targeting nuclear DNA-binding proteins.

BCL2A1, a member of the BCL-2 anti-apoptotic protein family, is overexpressed in severalhematologic malignancies and solid tumors. To identify a novel inhibitor of BCL2A1, we initiated a DEL screen. This screen identified a promising molecular scaffold featuring a unique but ultimately intractable reversible covalent bond with a buried lysine. Leveraging early covalent fragment-type mass spectrometry-based assays, we developed a new covalent lead that irreversibly engages a critical cysteine.

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.

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.

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.