Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors
October 26th, 2015
We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.
The overall goal of the strategy is to generate an effective small molecule drug-like therapeutic through conversion of a peptide inhibitor of a protein protein interaction. This is achieved by establishing a structure activity relationship for the desired peptide protein interaction to understand which parts can be effectively replaced as a second step. Computational chemistry is used to identify viable fragment alternatives for the known peptide determinants, which can then be used to synthesize a library of fragment peptide hybrids.
Next, a library of hybrids is synthesized between these fragments and the truncated peptide in order to generate molecules that can be tested in an in vitro assay to identify molecules that are more drug-like and which recapitulate the activity of the native peptide. The results show more drug-like compounds can be identified using the methodology, and these represent effective starting points for drug discovery of inhibitors of protein protein interactions. So the main advantage of this technique over existing methods like high throughput screening is that protein protein traction are extremely challenging from a drug discovery perspective, and conventional methods do not work well.
The replace strategy involves the stepwise iterative approach and therefore may take longer or is more likely to be effective in the long run. Today I will demonstrate most of the procedures myself with some assistance from my associate, Dr.Sandra Craig, who will set up some of the reactions and procedures. Begin this procedure with validation of the docking protocol, followed by preparation of the receptor binding site and ligands as described in the text protocol to dock the ligands into the Cyclin a groove.
First, select the ligand fit routine from the receptor ligand interactions protocol set to the software, use the receptor and the prepared ligands as input for this protocol. For these runs, select PLP one as the energy grid. The energy grid used by the docking program is the force field for generating ligand receptor interactions and potential poses.
Plp one is peace wise linear potential, which specifically prioritizes hydrogen bonding interactions. Specify that poses be energy minimized and the number of generated poses as 10. Leave all the other parameters at the default values in the first instance.
Display poses in the visualizer program and then sort by descending values of the PLP one score. Use scoring functions such as plp, one to estimate the binding affinity of a docked ligand based on a candidate ligand pose, geometry and non-covalent interaction with the target receptor structure. Visually analyze the top 25%of the scored poses for super impossibility with the known capping group.
Also observe whether the pose has fulfilled interaction filters, which are set to include atom restraints that require intermolecular contacts known to be critical for binding and or are required to position the potential capping group in the correct geometry for amide bond formation. Finally, examine poses for visual complementarity, which is defined as having efficient filling of the binding pocket in a manner that is consistent with known structure activity. Relationships for synthesis of end capping groups, use all commercial starting materials, solvents and reagents and synthesize by conventional synthetic organic chemistry.
Perform thin layer chromatography or TLC on silica gel for monitoring reactions by first dissolving the starting material and reaction mixture in the mobile phase. Then spot the dissolved starting material and reaction mixture on the TLC plate using capillary tubes. Place the TLC plate into the chamber containing the mobile phase.
Once the mobile phase reaches 90%of the TLC plate, remove and air dry the plate. Use UV light to detect the starting material and reaction mixtures as visible spots. Then calculate the RF of each spot, which is the ratio of distance traveled by the spot and the mobile phase.
Next, place the reaction mixture in a separatory funnel and wash it with aqueous acid or base solution as appropriate. Collect the organic solvent and evaporate it in a rotary evaporator before drying. The crude product obtained under a vacuum dissolve 500 milligrams of crude product in three to five milliliters of suitable solvent and added to a silica or RP 18 sample it before drying under air, placed a sample it containing the crude product in silica, reversed phase snap cartridges.
Purify the crude material using automated high performance flash chromatography. Employing a snap 100 gram column with a gradient. Run over 15 column volumes.
Dry the purified product collected in solvent from flash chromatography using a rotary evaporator by evaporating all of the solvent to dryness. Then further dry the product under vacuum to remove all the residual solvents. Perform characterization of the purified product by NMR MS and analytical HPLC as described in the text protocol to synthesize end caped fragment, ligated inhibitory peptides or flips assemble.
End ed peptic compounds through standard solid phase synthesis methods. Activate five equivalents of the C terminal amino acid in 4.4 equivalents of HBTU in two milliliters of DIMETHYLFORMAMIDE or DMF for five minutes. Load the mixture onto rink resin using six equivalents of di isopropyl ethyl amine for one hour at room temperature.
Remove the FM protecting group from the C terminal residue using 20%INE in three milliliters of DMF for 10 minutes. Couple subsequent amino acids step by step at each step. Couple five equivalents of the next amino acid using six equivalents of DI isopropyl, ethyl amine, and 4.4 equivalents of HBTU in two milliliters of DMF for one hour.
At room temperature, apply wash cycles after amino acid coupling and FM D protection steps after peptide assembly. Couple N capping groups using six equivalents of DI isopropyl, ethyl amine, and 4.4 equivalents of HBTU in two milliliters of DMF for one hour at room temperature upon completion of peptide assembly. Treat the reaction mixture with two milliliters of the cleavage mixture overnight to remove side chain protecting groups and Cleve flips from the resin tritrate the resulting product with cold dathyl ether to precipitate and if necessary, concentrate in a rotary evaporator filter the resulting solid and dry under vacuum or freeze dry using a lyophilizer.
At this point, proceed to the fluorescence polarization binding assay as described in the text protocol for determination of competitive binding. The model structure of H-A-K-R-R-L-I-F bound to the cyclone binding groove is shown. The cycling groove consists of two hydrophobic pockets, a larger primary pocket and a smaller secondary pocket, which are bridged by acidic residues depicted in red.
Other important interactions include hydrogen bonding contacts of the peptide backbone and ion pairing interactions of the three basic residues of the peptide here, the binding mode of three five D-C-P-T-R-L-I-F is shown, displayed with hydrogen bonding interactions with side chain atoms of tryptophan two 17 and glutamine 2 54 docked. Poses of three representative and capping groups are shown with each of these compounds possessing hydrogen bonds with the interaction filter atoms of tryptophan two 17 and glutamine 2 54. As depicted by green dashed lines, the fennel subsistent makes Vander wall's interactions with the secondary hydrophobic pocket.
After watching this video, you'll have a better understanding on how to perform and apply the replace strategy, converting a known peptide inhibitor into a more drug-like molecule, which inhibits a protein protein traction, and is a starting point for therapeutic development.
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Overview
This article describes the REPLACE strategy for targeting protein-protein interactions, aiming to convert peptide inhibitors into drug-like molecules. The methodology combines synthetic and computational approaches to enhance drug discovery.
Key Study Components
Area of Science
- Drug discovery
- Protein-protein interactions
- Computational chemistry
Background
- Protein-protein interactions are challenging targets for drug discovery.
- Conventional methods like high throughput screening often fail in this area.
- Peptide inhibitors can serve as starting points for developing small molecule therapeutics.
- The REPLACE strategy aims to improve the drug-like properties of these inhibitors.
Purpose of Study
- To generate effective small molecule drugs from peptide inhibitors.
- To establish structure-activity relationships for peptide-protein interactions.
- To identify viable fragment alternatives for known peptide determinants.
Methods Used
- Establishment of structure-activity relationships.
- Computational chemistry for fragment identification.
- Synthesis of a library of fragment peptide hybrids.
- In vitro assays to test the drug-like properties of synthesized molecules.
Main Results
- More drug-like compounds were identified using the REPLACE methodology.
- These compounds represent effective starting points for drug discovery.
- The approach overcomes limitations of traditional high throughput screening methods.
Conclusions
- The REPLACE strategy enhances the potential for developing inhibitors of protein-protein interactions.
- This method provides a systematic approach to drug discovery.
- Future research can build on these findings to further improve therapeutic options.
Frequently Asked Questions
Executive Industry Relevance
The REPLACE strategy addresses a critical bottleneck in oncology drug discovery by enabling the conversion of peptide-based protein-protein interaction inhibitors into drug-like small molecules. This approach expands the targetable space for cyclin-dependent kinases (CDKs), which are frequently deregulated in cancer and represent high-value therapeutic targets. By improving the drug-like properties of peptide inhibitors targeting the cyclin binding groove, the method supports early-stage target validation and lead identification efforts in oncology pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by validating the cyclin binding groove as a druggable site for selective CDK inhibition.
- Operational Value: Provides a systematic workflow for structure-activity relationship mapping to identify key residues amenable to replacement with fragment-like moieties.
- Predictive Value: Generates starting compounds with improved drug-likeness that recapitulate peptide inhibitor activity, supporting preclinical de-risking of CDK2/cyclin A interactions.
Screening & Assay Development
- Scientific Value: Produces fragment peptide hybrid libraries suitable for in vitro fluorescence polarization binding assays to quantify competitive binding at the cyclin binding groove.
- Operational Value: Establishes a reproducible synthesis and purification protocol using standard organic chemistry techniques, enabling scalable generation of analogs.
- Assay Readiness: Delivers compounds with defined binding poses and interaction filters that can be used to establish robust, quantitative screening assays for downstream hit-to-lead campaigns.
Translational & Preclinical Research
- Translational Value: Supports progression from biochemical inhibition to cellular context by yielding compounds that inhibit cell cycle CDK/cyclin complexes, linking target engagement to phenotypic outcomes.
- Preclinical Alignment: Focuses on non-ATP competitive inhibition, offering a mechanistic avenue to avoid kinase domain selectivity challenges associated with ATP-competitive CDK inhibitors.
- Risk Mitigation: Enables early assessment of binding mode conservation and interaction fingerprints, reducing uncertainty in target engagement prior to cellular efficacy testing.
Pipeline & Workflow Integration
The REPLACE strategy fits within the early discovery continuum, bridging peptide-based target validation and small molecule lead generation for oncology targets like CDKs.
- Discovery Biology: Supports hypothesis-driven validation of protein-protein interfaces by converting peptide inhibitors into synthetically tractable, drug-like molecules.
- Screening: Enables assay-ready compound libraries with measurable binding affinity and defined structure-activity relationships for hit identification.
- Analytics: Relies on docking scores, interaction filters, and visual complementarity assessments to prioritize compounds with favorable binding geometry and non-covalent interactions.
- Translational Research: Connects biochemical binding data to functional inhibition of CDK/cyclin complexes, supporting alignment with cell-based readouts.
- Enterprise Reuse: Establishes a modular, iterative framework applicable to other protein-protein interactions beyond CDKs, promoting platform-like utilization across discovery projects.
Operational & Enterprise Impact
- Scientific Value: Enhances target confidence by enabling structure-guided optimization of protein-protein interaction inhibitors with defined binding modes.
- Operational Value: Improves reproducibility through standardized computational docking, fragment library synthesis, and purification protocols.
- Strategic Value: Increases predictive confidence in early-stage projects by reducing reliance on empirical screening for challenging targets.
- Portfolio Impact: Facilitates risk-adjusted prioritization of CDK inhibitors by providing early chemical matter with validated target engagement and improved drug-like properties.
Implementation Considerations
- Requires expertise in computational chemistry for docking protocol validation and pose analysis using energy minimization and scoring functions.
- Depends on access to standard synthetic organic chemistry infrastructure for peptide assembly, fragment ligation, and purification via flash chromatography.
- Necessitates cross-team alignment between modeling, synthesis, and assay teams to ensure consistency in fragment selection and hybrid library design.
- Involves adaptation considerations when applying the strategy to different protein-protein interfaces, particularly in defining interaction filters and capping group geometries.
- Practical limitations include the iterative nature of the approach, which may require multiple cycles of design, synthesis, and testing to achieve optimal drug-like properties.
Biopharma FAQs
Chapters in this video
0:05
Title
1:57
Computational Identification of Potential Small Molecule Capping Groups
4:03
Synthesis of Potential N-capping Groups
6:25
Solid Phase Synthesis for the Generation of FLIPs
8:45
Results: Identification of More Drug-like Inhibitors of CDK2 through the Cyclin Groove
9:59
Conclusion
