Biology subjects

Ciulli, A.

Publications and source records attributed to Ciulli, A..

23 records · Page 2Linked to original sources

bioRxiv2023

Targeting cancer with small molecule pan-KRAS degraders

Despite the high prevalence of cancers driven by KRAS mutations, to date only the G12C mutation has been clinically proven to be druggable via covalent targeting of the mutated cysteine amino acid residue (1). However, in many cancer indications other KRAS mutations, such as G12D and -V, are far more prevalent and small molecule concepts that can address a wider variety of oncogenic KRAS alleles are in high clinical demand (2). Here we show that a single small molecule can be used to simultaneously and potently degrade 13 out of 17 of the most prevalent oncogenic KRAS alleles, including those not yet tractable by inhibitors. Compared with inhibition, degradation of oncogenic KRAS results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines. As a result, KRAS degraders inhibit growth of the majority of cancer cell lines driven by KRAS mutations while sparing models without genetic KRAS aberrations. Finally, we demonstrate that pharmacological degradation of oncogenic KRAS leads to tumour regression in vivo. Together, these findings unveil a new path towards addressing KRAS driven cancers with small molecule degraders. One-Sentence SummaryThe most prevalent KRAS variants which drive tumour growth in a major share of cancer patients can be targeted with a single small molecule degrader.

pharmacology and toxicology↗

bioRxiv2023

An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

Targeted protein degradation is a pharmacological modality based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via bifunctional compounds (PROTACs) composed of two separate warheads that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Using orthogonal genetic screening, biophysical characterization, and structural reconstitution, we investigate the mode of action of bifunctional BRD2/4 degraders (IBG1-4) and find that - instead of connecting target and ligase in trans as PROTACs do - they simultaneously engage two adjacent domains of the target protein in cis. This conformational change glues BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities which, albeit pre-existing, do not translate to BRD4 degradation in absence of compound. Structural insights into the ternary BRD4:IBG1:DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, termed intramolecular bivalent glues (IBGs), which work by bridging protein domains to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

biochemistry↗

bioRxiv2021

VHL inhibitor binding increases intracellular level of VHL

The von-Hippel Lindau (VHL) protein is a tumour suppressor protein frequently mutated in the VHL disease, which functions as substrate recognition subunit of a Cul2 E3 ubiquitin ligase (CRL2VHL). CRL2VHL plays an important role in oxygen sensing, by binding and targeting Hypoxia Inducible Factor-alpha subunits (HIF-alpha) for ubiquitination and degradation. VHL is also commonly hijacked by heterobifunctional degrader molecules known as proteolysis-targeting chimeras (PROTACs). In previous work we reported the structure-based design and functional characterisation of VHL inhibitors (VH032 and VH298) that induce the HIF response in cells. Here, we use unbiased quantitative mass spectrometry to identify the proteomic changes elicited by the VHL inhibitor and compare this to hypoxia or broad-spectrum prolyl-hydroxylase domain (PHD) enzyme inhibitor IOX2. Our results demonstrate the VHL inhibitor selectively activates the HIF response that is also present in the hypoxia- and IOX2-induced proteomic changes. Interestingly, VHL inhibitors were found to selectively upregulate a single protein, which is VHL itself. Our analysis revealed that this occurs via protein stabilisation of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn to reduced levels of HIF-1 protein. Our results demonstrate the high specificity of VHL inhibitors and suggest that use of these inhibitors would not produce overtly side effects due to prolonged HIF stabilisation. They also exemplify the concept that small-molecule binding induced protein stabilisation can increase protein levels inside cells.

biochemistry↗

bioRxiv2018

Structural insights into substrate recognition by the SOCS2 E3 ubiquitin ligase

The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. We solved cocrystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 A{square} and 2.69 A{square}, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, yet capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop, for the first time fully defined in the electron density, does not contact the substrate degrons directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.

biochemistry↗

bioRxiv2018

SPR-measured kinetics of PROTAC ternary complexes influence target degradation rate

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/451948v2_ufig2.gif" ALT="Figure 2"> View larger version (32K): org.highwire.dtl.DTLVardef@10e7c1eorg.highwire.dtl.DTLVardef@1f6a57forg.highwire.dtl.DTLVardef@1eb6e46org.highwire.dtl.DTLVardef@194cb2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗