The core of PROTAC molecular design lies in the precise construction of tri-functional molecules consisting of target protein ligands, E3 ligase ligands and linkers. Our design service adopts a multi-dimensional strategy: Target protein ligand screening: virtual screening based on target crystal structure databases (e.g. PDB), combined with molecular dynamics simulations to optimise binding sites. For targets lacking structural information, homology modelling and binding pocket prediction techniques are used. E3 ligase aptitude analysis: For common E3 ligases such as VHL, CRBN, etc., ligand libraries are established and tissue distribution specificity is evaluated. Optimisation of ligand binding free energy by quantum mechanical calculations. Ligand engineering: Simulate PROTAC conformation by molecular docking to optimise ligand length (typically 8-12 carbon chains) and rigidity/flexibility balance. Introduction of polyethylene glycol (PEG) or cyclic structures to regulate solubility and cell permeability. The central role of molecular docking technology: docking simulations of target protein-ligand and E3 ligase-ligand, respectively, using AutoDock Vina, Schrödinger and other software to calculate binding energy (ΔG) and conformational stability. Special attention was paid to the formation probability of ternary complexes, and the free energy of complex binding was assessed by MM-PBSA method to provide a thermodynamic basis for linker optimisation. PROTAC In Vitro Evaluation: the cornerstone of functional validation The in vitro evaluation system focuses on degradation efficiency, selectivity and mechanism of action validation: Degradation Activity Detection: Quantitative analysis of the degradation level (DC50 value) of target proteins by Western blot, CETSA (cellular thermal shift assay), and exclusion of transcriptional level interference by qPCR. Selective validation: whole protein stability analysis using proteomics (e.g. TMT tag quantification) to confirm the off-target effect of PROTAC on non-target proteins. Mechanism of action studies: Validation of ubiquitin-proteasome-dependent pathway through competition experiments (e.g., over-addition of E3 ligand), proteasome inhibitor (MG132) treatment; Surface Plasmon Resonance (SPR) to detect the kinetics of ternary complex formation. Typical example: The PROTAC molecule designed by a customer for BRD4 showed DC50=3 nM and more than 100-fold selectivity for BRD2/3 in in vitro experiments, which verified the enhancement of spatial selectivity by linker rigidification. PROTAC High-Throughput Screening: Accelerating the optimisation of lead compoundsIn response to the bottleneck of large molecular weight (MW>800) and poor membrane permeability of PROTAC, the High-Throughput Screening platform provides a rapid iterative capability: Microfluidic microarray technology: integrated cell culture-drug treatment-fluorescence detection module, which can complete the cell permeability testing of 500+ PROTAC variants for cell permeability testing. DEL (DNA-encoded compound library) linkage: Construct a linker diversity library (with modules for sulfonamides, alkynes, etc.) to screen high binding candidate molecules by affinity selection-sequencing technology. AI-driven QSAR model: Train a machine learning model based on historical data to predict the degradation activity and ADME properties of PROTAC, increasing the screening success rate by 40%. PROTAC In Vivo Animal Model: A Key Bridge to Translational Medicine Animal model research needs to address PROTAC-specific pharmacokinetic challenges: Pharmacokinetic (PD) model: adopt transplanted tumour mouse model (e.g. HCC1954 breast cancer), quantify the level of degradation of the target proteins in the tumour tissue by immunohistochemistry, and establish the PK/PD correlation with the plasma concentration. Correlation. Cross blood-brain barrier (BBB) assessment: To target neurodegenerative diseases, hCMEC/D3 cell BBB model was constructed in vitro, and the penetration efficiency was verified by cerebrospinal fluid sampling. Toxicity early warning system: physiological indicators of E3 ligase knockout mice were monitored to assess the effects of long-term degradation on normal tissues. For example, CRBN-dependent PROTAC needs to be excluded for teratogenic risk (thalidomide analogue). V. Synergistic innovation of technology modules In the whole process from molecular design to preclinical research, each module forms a closed-loop feedback: In vitro degradation data guides the optimisation of the linker, and the polarity of the molecule is lowered (cLogP<5) in order to improve the membrane permeability; The metabolic stability data from animal models are fed back to the design process, and deuterated or fluorinated modifications are introduced to extend the half-life; High-throughput screening is combined with AI prediction, to realise the "design-synthesis-test-analysis" process. The combination of high-throughput screening and AI prediction accelerates the ‘design-synthesis-test-analysis’ cycle. Prospects and Challenges Although PROTAC technology has made breakthroughs (e.g. ARV-471 entered phase III clinic), issues such as tissue-specific delivery and drug resistance mechanism still need to be solved. In the future, through the development of novel technologies such as dual E3 ligase aptamers and light-controlled PROTAC, combined with chemical proteomics and single-cell sequencing, it is expected to further expand the boundaries of its application.
