DEVELOPMENT OF A MOLECULAR FRAGMENT LIBRARY FOR ENZYME LIGAND IDENTIFICATION BY NMR

Fragment-Based Drug Design (FBDD) originated from the “SAR by NMR” method [1] and has been successfully employed by the pharmaceutical industry. Vemurafenib, developed by Plexxicon for the treatment of melanoma, was the first approved drug using the FBDD approach. This strategy has gained prominence in pharmaceutical development, leading to the approval of drugs such as sotorasib (Lumakras) and asciminib (Scemblix^®), among others, currently in clinical trials. FBDD uses fragment libraries to identify promising compounds as starting points for the development of new drugs. Fragments are chemical compounds with molecular weights under 300 Da, low binding affinity for the target, and low structural complexity. Ligand fragment identification can be performed using several methods, including differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography. However, academic research faces limitations, including the high cost of commercial libraries, low fragment quality (e.g., purity and chemical identity), and inadequate physicochemical properties for binding assays, such as poor solubility ^[2]. In this context, creating an in-house fragment library enables the identification of ligands and the generation of new prototypes for various targets. This project aims to generate a molecular fragment library to identify ligands for different enzymes. The initial compound selection was performed using the compound collection from the Laboratory for the Evaluation and Synthesis of Bioactive Substances (LASSBIO/ICB), comprising 2,281 compounds. The software KNIME Analytics was used to develop an automated workflow—pipelines capable of processing large volumes of chemical data. Compounds were first filtered based on the Rule of 3, considering criteria such as molecular weight (MW < 300), number of hydrogen bond donors (≤ 3), number of hydrogen bond acceptors (≤ 3), and number of rotatable bonds (≤ 3), resulting in 64 compounds meeting all these criteria. These compounds were then evaluated using PERCEPTA software (ACD/Labs), considering parameters such as CLogP (≤ 3) and solubility, selecting compounds with solubility ≥ 0.11 mg/mL. After this second filtering step, 31 compounds were selected to proceed to the next phase of the study. These compounds will undergo experimental testing to confirm solubility. Initially, samples will be prepared in DMSO-d6 and analyzed by ¹H NMR to assess purity and confirm chemical structure. Additionally, the solubility of the compounds in phosphate buffer will be evaluated at concentrations ranging from 0.25 to 1 mM, also using ¹H NMR.