Periodic Reporting for period 1 - KLKs4OvCa (Targeting human kallikreins involved in ovarian cancer pathogenesis: novel activity based probes and kallikrein-based therapeutic strategies.)
Berichtszeitraum: 2021-10-04 bis 2023-10-03
Aberrant protein ubiquitination and degradation are linked to the pathogenesis of several diseases including cancer, neurodegeneration, and metabolic diseases, that would benefit from targeted protein stabilization (TPS). Heterobifunctional molecules able to induce targeted protein deubiquitination (deubiquitinase targeting chimeras, DUBTACs) via engagement of a deubiquitinase (DUB) to a protein of interest (POI), can be recruited to stabilize proteins and restore their function (see figure). The identification of suitable DUB ligands has proven challenging and remain to be thoroughly explored. To overcome this limitation, we aimed to use covalent ligand discovery to explore novel DUBs for targeted protein stabilization. We established the following objectives:
1. WP1 (Objective 1): Development of a high-throughput screening platform for the discovery of covalent ligands for different effector proteins, using Ubiquitin C terminal hydrolase L1 (UCHL1) as model effector protein.
2. WP2 (Objective 2): Identification and development of selective ligands for UCHL1 targeting non catalytic cysteines through covalent fragment screening.
3. WP3 (Objective 3): Synthesis of UCHL1 DUBTACs and elucidation of mechanism of action (MoA) on protein stabilisation and ubiquitin biology.
1. WP1 (Objective 1): Development of a high-throughput screening platform for the discovery of covalent ligands for different effector proteins, using Ubiquitin C terminal hydrolase L1 (UCHL1) as model effector protein.
2. WP2 (Objective 2): Identification and development of selective ligands for UCHL1 targeting non catalytic cysteines through covalent fragment screening.
3. WP3 (Objective 3): Synthesis of UCHL1 DUBTACs and elucidation of mechanism of action (MoA) on protein stabilisation and ubiquitin biology.
WP1 (Objective 1): Covalent DUB recruitment enables the targeting of shallow pockets and allosteric ligandable hotspots, preserving DUB activity. Cysteine (Cys) amino acid can be targeted by electrophile warheads in covalent ligands. Most DUBs contain a Cys residue in their catalytic center, that needs to be preserved intact to maintain activity.
We identified two potential ligandable Cys residues on UCHL1 (Cys 132 and Cys 152). To study the ligandability of Cys 132 and Cys 152, we expressed UCHL1 mutant proteins carrying Cys132 or Cys152 native, while the other reactive Cys of the protein were mutated. Next, I developed a biochemical assay suitable for screening of non-catalytic ligands for UCHL1, further applicable to any effector protein. I established a fluorescence polarization (FP) assay for Cys ligands to monitor changes in Cys occupancy. The result, validated by Thermal shift assay (TSA) as well as mass spectrometry (MS) techniques, confirmed the suitability of this assay to identify ligands for a Cys residue of interest. The results from these assays evidenced that the Cys residue Cys132 was not reactive enough; therefore, we focused our effort to identify specific ligands for the Cys152 on UCHL1. Of note, the assay was successfully applied to other Cys residues in different effector proteins.
WP2 (Objective 2): I further optimized the assay from WP1 to a HTS format to reduce the time, costs, and to increase the reproducibility. I screened a library of 8000 Cys reactive compounds, and I identified approximately 200 compounds able to label Cys 152 ≥ 30% at 10 µM. To select the most promising ligands, I filtered the compounds based on different criteria, including i) validation of compound potency at different concentrations and incubation time, ii) removal of promiscuous compounds that showed activity against other Cys residues, iii) removal of compounds that would inhibit UCHL1 activity, iv) chemical tractability. I selected 34 compounds, and resynthesized representative examples of them. Next, I retested them in the FP assay to confirm the activity, and I also confirmed their binding to UCHL1 via MS. Successfully, most of the results for the resynthesized compounds were reproduced. To elucidate the in-cell target engagement of newly identified ligands, and the selective over other cysteine residues present in UCHL1, I developed an alkyne probe for a hit compound (PM38) to use in proteomics studies via click chemistry. The results obtained in two cell lines showed that the alkyne probe penetrates cells and binds to different proteins, including UCHL1. I further delved into the specific binding of the hit compound on UCHL1 using WT and mutant proteins. Preliminary results indicate that the compound can bind UCHL1 via Cys152, but further experiments are required to fully understand its mechanism of action.
WP3 (Objective 3): Lastly, I aimed to develope a small library of DUBTACs consisting of a UCHL1 binding moiety, a linker and a POI binding moiety. Based on the results from the HTS and SAR established, I was able to identify an exit vector in the hit compound PM38. Compound ESG252 consisted of a UCHL1 ligand, a linker, and a dTag recruiting molecule, that allows to recruit recombinant dTag fusion proteins (see figure). I engineered a recombinant mCherry-dTag fluorescent protein, to transfected HEK293T cells, and a subset of cells was treated with compound ESG252. Compound ESG252 can bind simultaneously to UCHL1 (via the PM38 moiety) and our POI mCherry (via the dTag moiety), bringing both proteins in close proximity. As result, the treatment with compound ESG252 leads to an increase in mCherry fluorescence, indicating that when UCHL1 is recruited to an mCherry protein, it leads to its stabilisation. We are currently exploring the mechanism of action of UCHL1 mediated protein stabilisation. Additionally, we are extrapolating these results to a pathologically relevant POI to further explore the potential of UCHL1 DUBTACs development.
We identified two potential ligandable Cys residues on UCHL1 (Cys 132 and Cys 152). To study the ligandability of Cys 132 and Cys 152, we expressed UCHL1 mutant proteins carrying Cys132 or Cys152 native, while the other reactive Cys of the protein were mutated. Next, I developed a biochemical assay suitable for screening of non-catalytic ligands for UCHL1, further applicable to any effector protein. I established a fluorescence polarization (FP) assay for Cys ligands to monitor changes in Cys occupancy. The result, validated by Thermal shift assay (TSA) as well as mass spectrometry (MS) techniques, confirmed the suitability of this assay to identify ligands for a Cys residue of interest. The results from these assays evidenced that the Cys residue Cys132 was not reactive enough; therefore, we focused our effort to identify specific ligands for the Cys152 on UCHL1. Of note, the assay was successfully applied to other Cys residues in different effector proteins.
WP2 (Objective 2): I further optimized the assay from WP1 to a HTS format to reduce the time, costs, and to increase the reproducibility. I screened a library of 8000 Cys reactive compounds, and I identified approximately 200 compounds able to label Cys 152 ≥ 30% at 10 µM. To select the most promising ligands, I filtered the compounds based on different criteria, including i) validation of compound potency at different concentrations and incubation time, ii) removal of promiscuous compounds that showed activity against other Cys residues, iii) removal of compounds that would inhibit UCHL1 activity, iv) chemical tractability. I selected 34 compounds, and resynthesized representative examples of them. Next, I retested them in the FP assay to confirm the activity, and I also confirmed their binding to UCHL1 via MS. Successfully, most of the results for the resynthesized compounds were reproduced. To elucidate the in-cell target engagement of newly identified ligands, and the selective over other cysteine residues present in UCHL1, I developed an alkyne probe for a hit compound (PM38) to use in proteomics studies via click chemistry. The results obtained in two cell lines showed that the alkyne probe penetrates cells and binds to different proteins, including UCHL1. I further delved into the specific binding of the hit compound on UCHL1 using WT and mutant proteins. Preliminary results indicate that the compound can bind UCHL1 via Cys152, but further experiments are required to fully understand its mechanism of action.
WP3 (Objective 3): Lastly, I aimed to develope a small library of DUBTACs consisting of a UCHL1 binding moiety, a linker and a POI binding moiety. Based on the results from the HTS and SAR established, I was able to identify an exit vector in the hit compound PM38. Compound ESG252 consisted of a UCHL1 ligand, a linker, and a dTag recruiting molecule, that allows to recruit recombinant dTag fusion proteins (see figure). I engineered a recombinant mCherry-dTag fluorescent protein, to transfected HEK293T cells, and a subset of cells was treated with compound ESG252. Compound ESG252 can bind simultaneously to UCHL1 (via the PM38 moiety) and our POI mCherry (via the dTag moiety), bringing both proteins in close proximity. As result, the treatment with compound ESG252 leads to an increase in mCherry fluorescence, indicating that when UCHL1 is recruited to an mCherry protein, it leads to its stabilisation. We are currently exploring the mechanism of action of UCHL1 mediated protein stabilisation. Additionally, we are extrapolating these results to a pathologically relevant POI to further explore the potential of UCHL1 DUBTACs development.
Results from this proposal are revealing, for the first time, the role of UCHL1 in targeted protein stabilisation that can be explored in pathologies related to abnormal protein degradation, and more generally can be exploited as a chemical biology tool in drug discovery. This proposal has also delivered a versatile technology platform to identify non-catalytic covalent ligands for UCHL1 that were not previously explored; this platform can be generally applicable to discover covalent ligands for numerous effector proteins of interest for induced proximity pharmacology, in a high throughput, fast and reproducible manner.
Over the last decade, there has been an ever-increasing interest in the development of heterobifunctional molecules for proximity induced pharmacology, however, their development is limited by the absence of rational and systematic approaches, and the reduced number of ligands available to recruit effector proteins. Our proposal pursued the identification of covalent ligands via recruitment to Cys residues that are non-essential for protein function. We have developed a biochemical assay that can be readily optimised for HTS to identify ligand for different effector proteins. These results can be further translated to more complex biological samples by including state-of-the-art proteomics and mass spectrometry techniques, facilitating the discovery of ligands in a cellular milieu. We have developed proof-of-concept UCHL1 DUBTACs to promote the targeted protein stabilisation of a POI. DUBTACs are gaining interest owing to their application as chemical biology tools complimentary to the genetical manipulation, and have great potential in diseases driven by abnormal protein degradation or heterozygous inactivating mutations (e.g. diabetes, and cancer). Aa a result, the proximity induced pharmacology community around the world can benefit from our versatile platforms, and our results open the opportunity for collaborations including both, the academic and the industrial sectors.
No website has been developed for the project
Over the last decade, there has been an ever-increasing interest in the development of heterobifunctional molecules for proximity induced pharmacology, however, their development is limited by the absence of rational and systematic approaches, and the reduced number of ligands available to recruit effector proteins. Our proposal pursued the identification of covalent ligands via recruitment to Cys residues that are non-essential for protein function. We have developed a biochemical assay that can be readily optimised for HTS to identify ligand for different effector proteins. These results can be further translated to more complex biological samples by including state-of-the-art proteomics and mass spectrometry techniques, facilitating the discovery of ligands in a cellular milieu. We have developed proof-of-concept UCHL1 DUBTACs to promote the targeted protein stabilisation of a POI. DUBTACs are gaining interest owing to their application as chemical biology tools complimentary to the genetical manipulation, and have great potential in diseases driven by abnormal protein degradation or heterozygous inactivating mutations (e.g. diabetes, and cancer). Aa a result, the proximity induced pharmacology community around the world can benefit from our versatile platforms, and our results open the opportunity for collaborations including both, the academic and the industrial sectors.
No website has been developed for the project