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CoAs / results for clinical and non-clinical lots |
Characterization of impurities that are unique to the drug product; under what conditions are they formed |
Justification for each specification |
Reference Standard CoA or summary of results |
If details are not provided in the batch records, documentation describing all components of the drug product package |
Stability Protocol(s) and details of study (e.g. packaging used for stability samples); Stability Data |
GPR132 RESEARCH PLAN |
CONFIDENTIAL: CONTAINS HEPTARES PLATFORM CONFIDENTIAL INFORMATION |
I) Outline of research plan |
General information & Deliverables |
Sosei Heptares will enter into a research collaboration agreement with Abbvie with the aim of developing an oral GPR132 small molecule agonist IND-ready Clinical Candidate for the treatment of IBD |
GPR132 Agonist Program |
Research Program Details |
Sosei Heptares' in-house work will concentrate on computational chemistry, medicinal chemistry, in vitro pharmacology, DMPK, stabilised GPCR (StaR protein) generation and structural biology to enable the generation of one Clinical Candidate. |
Primary screening (native conformation GPCR expressed by a cell, StaR protein binding and functional assays) and appropriate secondary screening (selectivity assays, etc.) will be performed by Sosei Heptares. Other activities outlined in this research plan will be outsourced through existing vendors or identification o... |
Estimated Timeline of Research Activities Performed by Sosei Heptares and Abbvie |
[Table showing timeline of activities over Q1-Q20] |
Platform |
a) StaR protein generation |
Thermostability assays will be established for the GPR132 receptor consistent with their use for agonist discovery, and Sosei Heptares will use its proprietary StaR protein and SABRE technology to identify thermostabilising mutations. These will be combined in an iterative way to shift the GPR132 receptor gradually to ... |
Deliverable: The sequences of stabilised receptors suitable for biophysical screening and structure determination with increased thermostability by ≥ 10C. |
b) StaR protein expression and purification |
GPR132 StaR proteins will be expressed in insect cells using the baculovirus system. A variety of approaches, such as a range of tags, codon optimisation, additives, enzyme cleavage and further mutagenesis, will be investigated in order to optimise the levels of receptor expression and to obtain homogeneous protein. Fo... |
Deliverable: Stabilised GPR132 receptor (>90% purity) suitable for biophysical screening and structure determination. QC by SDS PAGE, gel filtration and mass spec. |
c) Biophysics |
The primary platform for biophysical studies at Sosei Heptares will be Surface Plasmon Resonance (SPR) in which the GPR132 StaR protein or SuperStaR protein is immobilised to a sensor chip through a His tag. The affinities and kinetics of binding of tool agonists (or antagonists), and agonists (or antagonists) generate... |
Deliverable: Information on the affinities, kinetics and binding poses of compounds used to refine models of binding to the receptor and assist with compound optimisation along with homology models used to propose experiments and interpret results. |
d) Structure determination |
The GPR132 SuperStaR protein will be utilised for structure determination. Crystallisation trials will be set up for several constructs using a wide range of detergent conditions. These will include standard crystallisation screens as well as proprietary Sosei Heptares screens designed for GPCR crystallisation. Crystal... |
A crystal structure of the receptor should meet the following criteria: |
Data Statistics/Resolution: Maximum resolution of greater than, or equivalent to, 3.5Ä; where this is defined as a CC1/2 of not less than 0.5 with an I/σ (Mean((I)/sd(I)) equivalent to, or greater than, 1.0 in the outer (highest) resolution shell. Overall, the final dataset should have an associated Rpim of not more th... |
Model Building and Refinement: The final statistically validated atomic model for the target molecule should have associated and appropriate Rwork / Rfree values and ratios for the Resolution of the diffraction data as defined by: Rfree and the Rfree ratio according to: Derivation of expected values of cross-validation... |
A Cryo-EM structure of the receptor should meet the following criteria: |
Resolution in the region of the ligand binding site of at least 3.80 Å at an FSC figure of 0.143, in line with the principles described in Rosenthal P.B. & Henderson, R. J. Mol. Biol. (2003), 333: 721-745. The structure maps should be as good or better than an X-ray structure of the same resolution, with good definitio... |
Deliverable: Raw data, coordinates, mtz files for structures (x-ray and Cryo-EM). |
Drug Discovery |
a) Target Validation |
Demonstrate that the target is directly involved in a disease/mechanism process, and that engagement of the target will modulate its function and is likely to have a therapeutic effect. The following criteria are described to provide a framework for a multi-validation approach. Not all the following criteria described ... |
1. Preliminary target safety assessment |
a. In silico/literature review of target and known/predicted safety considerations |
2. Genetic association and Bioinformatics analysis |
a. Determine whether there is a genetic association/connection with the disease (e.g. eQTL/GWAS/transcriptomics) |
b. Provide an bioinformatic/systems biology overview of the pathway linking the target with a disease associated pathway/mechanism(s) |
3. In vitro cell-based primary/native assay data package |
a. Identify disease/mechanism relevant primary/native cells |
b. Demonstrate a clear and substantial effect on key cell process(es) that are relevant for (a) the target (e.g. 2nd messenger activation) and (b) disease relevant endpoints (e.g. proliferation, migration, invasion, cytokine secretion, tight junction formation etc) |
c. Confirm expression of target (mRNA / IHC) and effects using either a small molecule tool or antibody and/or knockdown/knockout using RNAi/CRISPR approaches |
d. Ideally effects should be evident in >1 cell line |
4. Translation feasibility assessment (to be performed in collaboration with Abbvie who will be responsible for running any chronic disease model studies, including those to provide tissue samples and if appropriate to support acute PK/PD target engagement) |
a. Provide an initial translational plan incorporating an early view on appropriate in vivo model(s) and biomarkers to support the drug discovery campaign |
b. Confirm target protein is expressed or active in the desired organ/subregion/cell types in a relevant 'disease model' |
c. Confirmation of an initial PKPD relationship and potential for demonstration of signals of efficacy in a relevant disease model with a tool compound/other means (on the assumption that tool compounds possess properties suitable for such experiments) |
Deliverable: Evidence that the GPR132 target increases or decreases in vitro pathways and increases or decreases in vivo PD assessments relevant to the target mechanism. |
b) Computational Chemistry: Modelling of GPR132 receptor structure |
Homology models of the GPR132 receptor with a range of different literature agonists (Appendix F) bound will be generated based on the published X-ray structures of analogous receptors and in-house data on relevant structures, guided by SAR, receptor mutation data, structural chemogenomics hypotheses (Brown (2019) PRP,... |
Structural cheminformatics and chemogenomics databases and analysis tools (e.g. de Graaf et al. ChemMedChem, 2018, 13, Tr Pharmacol Sci 2018, 39) will be used to: i) combine public and proprietary GPCR structure and ligand bioactivity data to enable the identification of structure-activity/function/selectivity relation... |
The models and ligand docking poses will be refined based on reported literature tools/SAR for GPR132 and any putative GPR132 binding site data reported, and structural chemogenomics hypotheses (Brown et al. (2019) PRP https://doi.org/10.1002/prp2.542 ). Thermostabilisation data generated during the GPR132 agonist StaR... |
The receptor homology models will be evaluated, prioritised, challenged, and optimised in an iterative manner based on: |
1. Their ability to rationalise and predict ligand SAR (e.g. explain importance of specific chemical groups, conformational properties, enable the discrimination of active/inactive molecules), and/or other pharmacological/biophysical experimental data (e.g. effects of mutations ligand binding/pharmacology should be con... |
2. Their applicability for the prospective identification (virtual screening), structure-based design and optimisation of novel ligands |
Models of the agonist (or antagonist) form of the receptor may be used to perform in silico screens of compound databases. Hits from screening will be docked into the models to help guide medicinal chemistry to elaborate hits. Sosei Heptares has developed and successfully applied combined energy-based and structural in... |
Molecular interaction field (GRID) and water interaction network (waterFLAP, waterMAP) based binding site analysis methods, Free Energy Perturbation (FEP+), and advanced MD methods/MetaMD have been developed at Sosei Heptares (in collaboration with Molecular Discovery and Schrodinger) to identify and prioritise druggab... |
To identify ligand induced and/or cryptic/transient allosteric binding pockets in GPR132, combined Molecular Dynamics (MD) and druggable binding site analysis methods have been developed at Sosei Heptares to identify (cryptic) druggable allosteric ligand binding pockets. These druggable allosteric GPR132 binding pocket... |
Deliverable: Models of the GPR132 receptor with the hits of the ≥ 2 chemical series including lead compounds bound consistent with known SAR. WaterMaps, MD trajectories, FEP+ maps with output G correlations. |
c) Stage 1: Hit identification |
The aim of the hit identification phase will be to use models of the structure of GPR132 to generate new chemical matter to identify structurally distinct hit series. The ≥ 2 chemically distinct series will have Tanimoto similarity values (based on ECFP4 fingerprint) of less than 0.4 to potentially de-risk off target a... |
Four approaches will be undertaken: |
1. Work-up and evaluation of current hits/tools reported from previous screening campaigns, utilising further compound design, synthesis and compound acquisition. |
Computational chemistry approaches, coupled with SDM, will provide binding site(s) and mode hypothesis to drive SAR optimisation and support hit confirmation via further file mining (internal and external) and/or early hit to lead chemistry. The ligand may act as a positive modulator that can be used to enhance acidic ... |
2. Scaffold hopping from existing compounds, using structural information / de novo design approaches from the refined model. |
Validated GPR132 ligand binding mode models in approach 1 can serve as templates for ligand and structure-based hit ID approaches in addition to virtual screening inspired (approach 3) to identify new scaffolds/ligands targeting the same binding site. Furthermore, biophysical mapping and crystallographic data can be us... |
3. Virtual screening based on the binding site(s) in an active (or inactive) state model of the receptor. |
Sosei Heptares will screen a database of commercially available compounds which are suitable starting points for drug discovery, selecting a subset of virtual hits for testing in assays. Complementary ligand-based and protein-based virtual screening approaches will be used including: - Ligand-based approaches will incl... |
GPCR structure/sequence analysis based chemogenomics approaches facilitated by Sosei Heptares' 3Decision enabled GPCR structural databases combining public and proprietary GPCR structural information and bio/chemoinformatics tools (including ss-Tea, BioGPS, and customised GPCR structural cheminformatics workflows) will... |
4. Screening of Sosei Heptares proprietary GPCR diversified libraries generated through a combination of chemoinformatic data mining and manual curation techniques |
Customised screening libraries designed to cover the GPCRome to enable early hit identification and establish target tractability. These will be profiled in a developed wild-type GPR132 agonist assay in vitro and/or via SPR (when GPR132 agonist StaR protein is available). Combinations of complementary chemical librarie... |
Single concentration hits from any of the approaches outlined above will be confirmed by re-testing in the same assay by profiling in concentration response to generate a pIC50/pKi/pEC50 result and checked with orthogonal assays if feasible. |
Additional Hit ID utilising StaR protein |
The aim of this phase will be to use GPR132 StaR protein to generate new chemical matter via enablement of the following potential approaches: |
1. GPR132 StaR protein will be characterised to confirm retainment of agonist binding to support hit identification using the thermostabilised receptor. Stabilised GPR132 will be purified in detergent to provide soluble protein and enable the potential application of DNA-encoded library screening against GPR132. GPR132... |
2. Fragment screening will be employed when a suitable biophysical assay format has been established for GPR132 using a focused fragment library. Based on experience of family A targets we might expect acidic fragments (which are well represented in the Heptares fragment library) to bind different orthosteric and allos... |
"Technical Failure" for GPR132 Agonist Program will mean that Heptares fails to deliver a completed GPR132 Stabilized Receptor (StaR protein) which is subsequently demonstrated to enable compound screening in e.g. SPR or DNA-encoded library platforms as described above in section 'b) Stage 1: Hit identification'. |
Stage 1 (Hit ID) Deliverable: Identification of ≥ 2 novel structurally distinct hit chemical series with calculated ECFP4 Tanimoto similarity values of less than 0.4 and on target potency <10uM in a GPR132 overexpressing cell line with cAMP readout and less than 3x standard deviation of vehicle control in parental cell... |
c) Stage 2: Lead Generation |
Aim: To identify lead candidates from 2 distinct chemical series with the calculated ECFP4 Tanimoto similarity values of less than 0.4 and demonstrate in vitro and acute in vivo activity |
• To identify potent modulators of GPR65 using SAR/SBDD medicinal chemistry efforts. |
• To evaluate ADME/PK properties of selected hits. |
• To demonstrate efficacy in an acute in vivo model with proprietary compound(s). |
Once hits are confirmed and concentration response curves obtained, the most promising examples for further elaboration will be selected based on several factors, including molecular efficiencies, synthetic tractability, confidence in modelled binding modes, and prioritising chemotypes based on developability. |
Complementary 2D/topology based (fast GPU similarity, orthogonal molecular fingerprints) and 3D based similarity search methods (shape/pharmacophore search, protein docking-structural interaction fingerprint scoring protocols) against purchasable, synthesisable (e.g. Enamine REAL), and de novo virtual library design ap... |
Complementary medicinal chemistry ligand design strategies will be used in conjunction with above hit elaboration approaches, including structure-based bio-isosteric replacement strategies based on comparative analyses of combined public and in-house GPCR structures and models. Physiochemical property assessment in com... |
Binding poses for key molecules will be obtained from docking into models, enhanced with Biophysical Mapping data if feasible, or, if possible, from X-ray structures. Molecule design will be part guided by docking poses, and part empirically to build up SAR of the series. As more SAR is established, and confidence incr... |
As described in section b), experimentally enhanced GPR65 models accommodating different orthosteric and allosteric ligand binding sites and agonist/antagonist conformations are available based on public and proprietary insights into class A GPCR structure, biophysical (e.g. HDX), and pharmacological (mutation mapping)... |
The in vitro pharmacology of compounds will be tested in primary functional assays to determine potency/efficacy and competition binding if available for affinity measurements. Activity at orthologue receptors will also be examined to understand cross species activity in addition to other off-target GPCR selectivity as... |
Molecules with appropriate pharmacology and in vitro ADME properties will be selected for rodent PK profiling to establish IVIV Cl correlation and oral bioavailability determination. Molecules with sufficient free exposure cover over primary pharmacology will be further selected as tools for in vivo efficacy profiling ... |
Stage 2 (Lead Generation) Deliverable: To identify lead molecules from two (2) prioritised and distinct chemical series with appropriate pharmacology, ADME, PK and in vivo acute PK/PD consistent with the criteria in Appendix B. Translational biomarker strategy will be generated to outline clinically feasible biomarker ... |
d) Stage 3: Lead Optimisation |
Aim: To optimize lead candidates from two (2) distinct chemical series and demonstrate in vivo activity in a relevant chronic disease model(s) and acceptable safety profile |
• To demonstrate efficacy in appropriate in vivo disease model(s) |
The prioritised and elaborated two (2) lead chemical series will be subjected to further optimisation. Both binding affinity and functional activity of compounds will be monitored, with the emphasis on relevant assay(s) that correlate to PK/PD understanding. Parameters such as residence time on the receptor may also be... |
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