Biology subjects

Gazgalis, D.

Publications and source records attributed to Gazgalis, D..

4 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure and biochemical characterization of a BRAF/CRAF heterodimer: Negative charge in the NtA motif is not required for RAF activation

Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero-dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.

biophysics↗

bioRxiv2025

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

biochemistry↗

bioRxiv2025

Overcoming EGFR resistance by monovalent and bident inhibitors targeting Cys775

Covalent targeting of EGFR cysteine 797 by osimertinib is one of the most successful breakthroughs in targeted therapy, fundamentally transforming the treatment landscape for non-small cell lung cancer (NSCLC) patients. However, resistance driven by mutation of C797 remains a major clinical challenge. Developing novel covalent strategies beyond C797 targeting presents a compelling opportunity for next-generation EGFR inhibitors. We first demonstrated that cysteine 775, located deep within the ATP-binding pocket, is accessible by a rationally designed covalent molecule ZNL-3, which as the first-in-class covalent cysteine 775 inhibitor exhibited strong efficacy in osimertinib-resistant mouse models. To further enhance resilience to resistance-causing mutations, we developed a dual-warhead, bident compound--YNW-1--which covalently targets both cysteine 775 and 797 simultaneously. YNW-1 is the first intramolecular lock to exhibit balanced reactive efficiency on both cysteines, rendering single-site mutations ineffective to confer resistance. The discovery of ZNL-3 and YNW-1 represents significant advancements in EGFR-targeted drug development, and further optimization toward clinical translation is a worthwhile strategy. SIGNIFICANCE: This study establishes the therapeutic potential of an EGFR covalent inhibitor through unprecedented targeting of cysteine 775 and provides the first demonstration that dual cysteine engagement offers superior efficacy over conventional covalent inhibitors by delaying resistance.

cancer biology↗

bioRxiv2023

De novo design of drug-binding proteins with predictable binding energy and specificity

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low M, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles. One Sentence SummaryWe use informatic sampling to design low nM drug-binding proteins, and physics-based calculations to accurately predict affinities.

biophysics↗