Biology subjects

Kozicka, Z.

Publications and source records attributed to Kozicka, Z..

5 recordsLinked to original sources

bioRxiv2025

A functional map of CDK-drug interactions at single amino acid resolution

Proteins that drive or support human disease phenotypes are attractive molecular targets for precision therapy, yet most are nominated by knockout studies and then targeted with drugs that inhibit core catalytic pockets. These strategies cannot resolve which residues are essential, whether non-catalytic sites offer better selectivity or potency, or identify on-target resistance mechanisms. We introduce a framework that integrates precision genome editing, mechanistically diverse therapeutics, and computational sequence-structure-function analysis to map protein essentiality and potential druggability at single amino acid resolution. Applying this framework across 9 cyclin-dependent kinases (CDKs) and 15 cancer therapeutics--including ATP-competitive inhibitors, PROTACs, and molecular glue degraders--we identify shared and CDK-specific residues critical for cell fitness and drug response, including known resistance mutations and dozens of new variants. The resulting functional maps resolve residue- and mechanism-specific differences in the resistance spectra among agents targeting the same protein. We show that this iterative strategy can also uncover higher order interactions by performing intra- and extragenic epistasis screens to identify residues that mediate on-target and within-family cell fitness and drug resistance. Finally, we find evidence of novel CDK6 mutations in breast cancer patients and concordance between experimental and clinical correlates of response to CDK4/6 inhibitors. By mapping residue-level essentiality and forecasting therapy resistance mutations, target-drug interaction maps could inform clinical treatment and guide design of more selective therapeutic molecules.

cancer biology↗

bioRxiv2025

Effective Tubulin Degradation by Rationally Designed Proteolysis Targeting Chimeras

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that induce the degradation of proteins of interest (POIs) via the ubiquitin-proteasome pathway by recruiting E3 ligases to form a ternary complex with the POI. In this study, we rationally designed and synthesized PROTACs targeting the {beta}-tubulin heterodimer, the building block of microtubules (MTs) that are essential for numerous cellular functions and represent important therapeutic targets in cancer and neurodegenerative diseases. Maytansinol, a known MT-destabilising agent, was selected as the POI ligand, functionalised and conjugated to linkers bearing cereblon or Von Hippel-Lindau ligands as E3 ligase recruiters. Four compounds were synthesized and characterized through structural, biophysical and cell biology studies to evaluate their ability to form degradation-prone tubulin-PROTAC-E3 ligase ternary complexes. We confirmed that the PROTACs effectively bind tubulin and recruit E3 ligases. Remarkably, two PROTACs exhibited cellular degradation activity, representing an important advancement in chemically inducing tubulin-E3-ligase interactions. This work integrates rational design, biophysical and structural validation, and cell-based studies to establish a robust framework for developing tubulin-targeting PROTACs, offering significant implications for basic research and therapeutic developments.

biochemistry↗

bioRxiv2024

Discovery of electrophilic degraders that exploit SNAr chemistry

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly undruggable targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

biochemistry↗

bioRxiv2024

Charged Molecular Glue Discovery Enabled by Targeted Degron Display

Small molecules that induce protein interactions hold tremendous potential as new medicines, as probes for molecular pathways, and as tools for agriculture. Explosive growth of targeted protein degradation (TPD) drug development has spurred renewed interest in proximity-inducing molecules and especially Molecular Glue Degraders (MGDs). These compounds catalyze destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for pre-defined targets is a major challenge in contemporary drug discovery. The field is limited by a lack of approaches that can exploit charged ligand-binding pockets, thus excluding a major fraction of ubiquitin ligases (E3s) that evolved to recognize exceedingly common acidic and basic degrons. Here we solve these important chemical challenges through "chemocentric" MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue (c-Glue). ZZ1 activation unmasks a sulfinic acid moiety that binds the modular GID/CTLH ubiquitin ligase complex via a basic pocket in its YPEL5 subunit. YPEL5 is a CRBN structural homolog and an essential non-Cullin ubiquitin ligase cofactor expressed in cancers of the bone marrow. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit GID/CTLH substrates, and they provide a powerful strategy to discover c-Glues that induce proximity to ubiquitin ligases with similarly desirable properties.

biochemistry↗

bioRxiv2022

Readout of histone methylation by Trim24 locally restricts chromatin opening by p53

The genomic binding sites of the transcription factor (TF) and tumour suppressor p53 are unusually diverse in regards to their chromatin features, including histone modifications, opening the possibility that chromatin provides context-dependence for p53 regulation. Here, we show that the ability of p53 to open chromatin and activate its target genes is indeed locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated lysine 4 of histone H3, thereby preferentially locating to those p53 sites that reside in closed chromatin, while it is deterred from accessible chromatin by lysine 4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to impact gene expression as a function of the local chromatin state. These findings link histone methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors which locally modulate TF function.

genomics↗