Periodic Reporting for period 1 - ProCenDecl (Synthesis and validation of chemical Probes for Centrosome Declustering: development of potent and selective anti-cancer agents.)
Reporting period: 2019-09-01 to 2021-08-31
Issue/problem addressed and importance for the society:
Breast cancer remains the second most common cause of cancer death in women in the UK, with around 11,400 deaths annually. Advances in treatment of primary tumours mean that over 85% of patients diagnosed at early stages will survive ≥5 years, but the occurrence of distal metastasis drops 5-year survival rates to <30% for stage IV patients and there is a pressing need for strategies to reduce recurrence of cancer following removal of primary tumours. Although drug-resistant metastasis is a major killer for many types of cancer and remains mostly uncurable, preventative strategies have slowly but steadily improved disease prognosis. Systemic treatment regimens following or preceding surgery (adjuvant or neoadjuvant therapy, respectively) show great promise for prevention of recurrence and metastasis, significantly decreasing mortality rates, particularly for breast cancer. The potential for significant gains for treatments targeting the early metastatic niche is highlighted by the recent success of adjuvant bisphosphonate therapy in breast cancer, which is expected to prevent ≥3 in every 100 deaths of breast cancer annually in the UK.
The main focus of the conducted research project was achieving Rab27a inhibition using novel selective and potent molecules. Rab27 inhibition is most likely to find clinical utility as part of an adjuvant or neoadjuvant treatment regimen in a patient population at significant risk of metastatic recurrence despite standard systemic therapy in the treatment of breast cancer. The initial focus on breast cancer is based on previously obtained preclinical data, significant unmet need, and a feasible clinical development pathway for this unconventional class of agents, but if successful it is likely that Rab27 inhibition could be similarly applied to other solid tumours. The proprietary preliminary data developed in the research group prior my arrival strongly supported the proposal that Rab27a inhibition would enhance the efficacy of adjuvant therapy by targeting the early metastatic niche. There were compelling evidence in models of post-surgery recurrence supporting Rab27a as a valid target in this context.
Overall objectives:
The conducted research had as main focus the development of Rab27a-selective macrocyclic peptide inhibitors with the potential to benefit patient prognosis by offering a novel approach for targeted adjuvant therapy with reduced treatment burden.
Based on preliminary data and extensive in-house assay platform to support development of Rab27a inhibitors, the main objectives for this project were:
1) Identify and develop first in class Rab27a macrocyclic peptide inhibitors;
2) Validate the efficacy of targeting Rab27a with peptide inhibitors using a range of biophysical assays (SPR, thermal shift, FP);
3) Confirm the efficacy of the novel peptide inhibitors in effectively disrupting the Rab27a-effector protein interactions;
4) Evaluate the in cell-permeability of the novel macrocyclic peptide inhibitors;
5) Develop chemical probes to test target engagement in relevant cell-lines.
Breast cancer remains the second most common cause of cancer death in women in the UK, with around 11,400 deaths annually. Advances in treatment of primary tumours mean that over 85% of patients diagnosed at early stages will survive ≥5 years, but the occurrence of distal metastasis drops 5-year survival rates to <30% for stage IV patients and there is a pressing need for strategies to reduce recurrence of cancer following removal of primary tumours. Although drug-resistant metastasis is a major killer for many types of cancer and remains mostly uncurable, preventative strategies have slowly but steadily improved disease prognosis. Systemic treatment regimens following or preceding surgery (adjuvant or neoadjuvant therapy, respectively) show great promise for prevention of recurrence and metastasis, significantly decreasing mortality rates, particularly for breast cancer. The potential for significant gains for treatments targeting the early metastatic niche is highlighted by the recent success of adjuvant bisphosphonate therapy in breast cancer, which is expected to prevent ≥3 in every 100 deaths of breast cancer annually in the UK.
The main focus of the conducted research project was achieving Rab27a inhibition using novel selective and potent molecules. Rab27 inhibition is most likely to find clinical utility as part of an adjuvant or neoadjuvant treatment regimen in a patient population at significant risk of metastatic recurrence despite standard systemic therapy in the treatment of breast cancer. The initial focus on breast cancer is based on previously obtained preclinical data, significant unmet need, and a feasible clinical development pathway for this unconventional class of agents, but if successful it is likely that Rab27 inhibition could be similarly applied to other solid tumours. The proprietary preliminary data developed in the research group prior my arrival strongly supported the proposal that Rab27a inhibition would enhance the efficacy of adjuvant therapy by targeting the early metastatic niche. There were compelling evidence in models of post-surgery recurrence supporting Rab27a as a valid target in this context.
Overall objectives:
The conducted research had as main focus the development of Rab27a-selective macrocyclic peptide inhibitors with the potential to benefit patient prognosis by offering a novel approach for targeted adjuvant therapy with reduced treatment burden.
Based on preliminary data and extensive in-house assay platform to support development of Rab27a inhibitors, the main objectives for this project were:
1) Identify and develop first in class Rab27a macrocyclic peptide inhibitors;
2) Validate the efficacy of targeting Rab27a with peptide inhibitors using a range of biophysical assays (SPR, thermal shift, FP);
3) Confirm the efficacy of the novel peptide inhibitors in effectively disrupting the Rab27a-effector protein interactions;
4) Evaluate the in cell-permeability of the novel macrocyclic peptide inhibitors;
5) Develop chemical probes to test target engagement in relevant cell-lines.
Novel macrocyclic peptides for Rab27a-effector inhibition (PPI):
1) A >10^12 member macrocyclic peptide mRNA display library screen against Rab27a (C123S/C188S double mutant, GTP-loaded), was performed in collaboration with Dr Louise Walport at the Crick Institute in London. The screen allowed us to identify novel peptide hits, 8 in particular, that showed a good binding affinity for Rab27a. These
cyclic peptides were synthesized and purified, then tested by SPR (Surface Plasmon Resonance) to check their binding affinity for the selected target. Among them only one, CP1, showed good binding properties as well as synthetic feasibility and tractability. CP1 was taken forward to other biophysical assays.
2) CP1 binding affinity was confirmed using another biophysical assay, Thermal Shift (DSF). The assay was previously optimized and adapted from a literature procedure.
3) The ability of CP1 in disrupting the protein-protein interaction was assessed using a Fluorescence Polarisation assay (FP). A TAMRA-analogue of a known Rab27a effector (Slp2a) was synthesized and used in the assay to assess the inhibitory activity of the peptide hit.
4) An alanine scanning was performed and allowed the discovery of a better peptide ligand, in terms of physical chemical properties, CP1[I10A] (Rab27a KD= 93nM).
5) CP1[I10A] was successfully crystallized with Rab27a (C123S/C188S double mutant) in its native form.
6) An in-cell peptide permeability assay (CAPA) using a chloroalkane tagged peptide in HeLa cells stably expressing a HaloTag-GFP construct was performed.
7) Photo-activatable analogues of CP1 were designed and synthesized and their in-cell activity addressed in MDA-MB 231 cells (triple negative breast cancer cell line).
1) A >10^12 member macrocyclic peptide mRNA display library screen against Rab27a (C123S/C188S double mutant, GTP-loaded), was performed in collaboration with Dr Louise Walport at the Crick Institute in London. The screen allowed us to identify novel peptide hits, 8 in particular, that showed a good binding affinity for Rab27a. These
cyclic peptides were synthesized and purified, then tested by SPR (Surface Plasmon Resonance) to check their binding affinity for the selected target. Among them only one, CP1, showed good binding properties as well as synthetic feasibility and tractability. CP1 was taken forward to other biophysical assays.
2) CP1 binding affinity was confirmed using another biophysical assay, Thermal Shift (DSF). The assay was previously optimized and adapted from a literature procedure.
3) The ability of CP1 in disrupting the protein-protein interaction was assessed using a Fluorescence Polarisation assay (FP). A TAMRA-analogue of a known Rab27a effector (Slp2a) was synthesized and used in the assay to assess the inhibitory activity of the peptide hit.
4) An alanine scanning was performed and allowed the discovery of a better peptide ligand, in terms of physical chemical properties, CP1[I10A] (Rab27a KD= 93nM).
5) CP1[I10A] was successfully crystallized with Rab27a (C123S/C188S double mutant) in its native form.
6) An in-cell peptide permeability assay (CAPA) using a chloroalkane tagged peptide in HeLa cells stably expressing a HaloTag-GFP construct was performed.
7) Photo-activatable analogues of CP1 were designed and synthesized and their in-cell activity addressed in MDA-MB 231 cells (triple negative breast cancer cell line).
Progress achieved and results:
1) Within this project, we have identified the first potent macrocyclic peptide ligands targeting native Rab27a, with activity against Rab27a-effector (Slp2a) protein-protein interactions (PPIs) and target engagement both biochemically (using recombinant Rab27a and MDA-MD 231 cell lysates) and in cells (MDA-MB 231 cell line); furthermore,
we have established an assay cascade which can efficiently progress compounds (SPR, DSF, FP). These major milestone outcomes support Rab27a as a tractable and ligandable target.
2) The peptide inhibitor CP1[I10A] showed a promising permeability in a CAPA assay (Chloro-alkane penetration assay), overcoming one of the main issues of peptides. Its metabolic stability has to be assessed if further in vivo studies will be performed.
3) We were the first one able to co-crystallize our peptide inhibitor with Rab27a in its native form. This is an indication that the macrocyclic peptide was able to considerably stabilize the protein in its native form and this was never observed before with other published ligands.
4) A photo-activatable probe analogue of CP1[I10A] was successfully synthesized and retained a good binding affinity for Rab27a. This analogue was able to photo crosslink the recombinant protein and target engagement experiments in intact cells are currently on-going.
As a future development of the project, the phenotypic effect of these inhibitors in relevant model systems of inflammation or cancer should be evaluated. The impact on the society could be relevant.
1) Within this project, we have identified the first potent macrocyclic peptide ligands targeting native Rab27a, with activity against Rab27a-effector (Slp2a) protein-protein interactions (PPIs) and target engagement both biochemically (using recombinant Rab27a and MDA-MD 231 cell lysates) and in cells (MDA-MB 231 cell line); furthermore,
we have established an assay cascade which can efficiently progress compounds (SPR, DSF, FP). These major milestone outcomes support Rab27a as a tractable and ligandable target.
2) The peptide inhibitor CP1[I10A] showed a promising permeability in a CAPA assay (Chloro-alkane penetration assay), overcoming one of the main issues of peptides. Its metabolic stability has to be assessed if further in vivo studies will be performed.
3) We were the first one able to co-crystallize our peptide inhibitor with Rab27a in its native form. This is an indication that the macrocyclic peptide was able to considerably stabilize the protein in its native form and this was never observed before with other published ligands.
4) A photo-activatable probe analogue of CP1[I10A] was successfully synthesized and retained a good binding affinity for Rab27a. This analogue was able to photo crosslink the recombinant protein and target engagement experiments in intact cells are currently on-going.
As a future development of the project, the phenotypic effect of these inhibitors in relevant model systems of inflammation or cancer should be evaluated. The impact on the society could be relevant.