Project Grant R44GM140666

A MICROED PIPELINE FOR THE PHARMACEUTICAL AND BIOTECHNOLOGY INDUSTRY - PROJECT SUMMARY/ABSTRACT MICROCRYSTAL ELECTRON DIFFRACTION (MICROED) IS AN EMERGING STRUCTURE DETERMINATION TECHNIQUE OF HIGH INTEREST TO THE PHARMACEUTICAL AND BIOTECHNOLOGY INDUSTRIES. MICROED CAN RAPIDLY DETERMINE ATOMIC-RESOLUTION STRUCTURES FROM MICROCRYSTALS WITH MINIMAL SAMPLE REQUIREMENTS. THE GOAL OF THIS PHASE II PROPOSAL IS TO DEVELOP AND DELIVER ROBUST SERVICE OFFERINGS FOR MICROED STRUCTURE DETERMINATION TO THE COMMERCIAL SECTOR. IMPROVEMENTS TO CRITICAL STEPS IN THIS WORKFLOW WILL ENABLE ANALYSIS FOR ANY SUITABLE SAMPLE TYPE WHILE OFFERING A LEVEL OF EFFICIENCY THAT MEETS THE REQUIREMENTS OF STRUCTURE-BASED DRUG DESIGN (SBDD) AND MEDICINAL CHEMISTRY PROGRAMS. THE PERMISSIVE SAMPLE REQUIREMENTS, MINIMAL TIME REQUIREMENTS, AND HIGH-QUALITY STRUCTURES GENERATED MAKE MICROED AN ATTRACTIVE STRUCTURE DETERMINATION TECHNIQUE WITH BROAD APPLICATIONS FOR SMALL MOLECULES, PEPTIDES, NATURAL PRODUCTS, PROTEINS AND PROTEIN-DRUG COMPLEXES. AN EFFICIENT MICROED PIPELINE WILL HELP TO REDUCE THE COST OF DRUG DEVELOPMENT AND REDUCE THE TIME IT TAKES A DRUG TO MAKE IT INTO THE CLINIC. MICROED USES A TRANSMISSION ELECTRON MICROSCOPE (TEM) TO COLLECT DIFFRACTION DATA FROM SMALL CRYSTALLINE SAMPLES. THE STEPS REQUIRED INCLUDE TRANSFERRING CRYSTALLINE SAMPLES TO TEM GRIDS; COLLECTING A SERIES OF CONTINUOUS-ROTATION, ELECTRON-DIFFRACTION DATASETS FROM SINGLE CRYSTALS; PROCESSING THE DIFFRACTION DATA AND REFINING THE ATOMIC MODEL. AN INITIAL DEVELOPMENT PHASE DEMONSTRATED COLLECTION OF EXCELLENT QUALITY DIFFRACTION DATASETS FOR MORE THAN 20 SAMPLES, AND THE ABILITY TO DELIVER ATOMIC RESOLUTION MAPS SUITABLE FOR ADDRESSING THE NEEDS OF OUR PHARMACEUTICAL CLIENTS WHO TOOK PART IN ALPHA AND BETA TESTING. NEW METHODS WILL BE DEVELOPED, AND EXISTING APPROACHES OPTIMIZED TO IMPROVE EVERY STEP OF THIS PIPELINE, SUBSTANTIALLY INCREASE THROUGHPUT, AND IMPROVE FINAL MAPS AND MODELS. SPECIFIC AIMS INCLUDE: (1) OPTIMIZING TEM GRID PREPARATION TO ENSURE THE TRANSFER OF A SUFFICIENT NUMBER OF HIGH-QUALITY TARGET MICROCRYSTALS ONTO THE GRID SUBSTRATE WITHOUT DAMAGING THEM OR ALTERING THEIR PROPERTIES. (2) DEVELOPING AN AUTOMATED, HIGH-THROUGHPUT DATA COLLECTION SYSTEM CAPABLE OF UNATTENDED, OVERNIGHT DATA ACQUISITION FOR MULTIPLE DIFFERENT SAMPLES, AND OF PROVIDING INFORMATION TO INFORM DATA SCALING AND DETERMINING HANDEDNESS OF CHIRAL SMALL MOLECULES. (3) AUTOMATING AND IMPROVING DATA PROCESSING, PHASING AND MODEL REFINEMENT INCLUDING REAL-TIME DATA REDUCTION, BRUTE FORCE ON-THE-FLY PHASING, AND INVESTIGATING A WIDE RANGE OF NEW PROGRAMS AND METHODS. IN PARALLEL WITH THE PHASE II RESEARCH PLAN, A FULL COMMERCIAL SERVICE OFFERING SPANNING THE COMPLETE RANGE OF MICROED CAPABILITIES WILL BE DEVELOPED.