Richard S. L. Stein CS 379a February 21, 2006

1. BREED: Generating Novel Inhibitors through Hybridization of Known Ligands(A. C. Pierce, G. Rao, and G. W. Bemis) Richard S. L. Stein CS 379a February 21, 2006

2. Examining Protein-Ligand Interactions • Comparison of more than a small number of protein-inhibitor complex structures is impossible manually • Hundreds of complex structures are available and more are being discovered constantly • Combining fragments of known ligands is a promising approach for finding new compounds • Manual recombination of fragments is tedious and can take prohibitively long

3. The Role of BREED • Automates the recombination of fragments of known ligands to produce new inhibitors • Generates a large number of new compounds from a small number of initial ligands • Applied to HIV-1 aspartyl protease and protein kinases

4. Methodology • All ligands are saved in a database with implicit hydrogen atoms • Ligands are examined pairwise to find matching bonds • Starting molecules are divided into two parts on either side of any matching bond • New compounds formed by mixing & matching parts of initial molecules • No bond contained in a ring may be used as a matching bond for this process • Above steps can be repeated to form further hybrid compounds

5. Initial and Generated Compounds from HIV-1 Protease Inhibitors

6. Activity of Newly Generated Ligands • Compound 5 was synthesized and found to have HIV-1 protease inhibitory activity (IC50 = 160 nM) • Compound 6 was synthesized with a modification (aniline  benzoxadiazole) and found to have high HIV-1 protease affinity (Ki = 0.1 nM) • A variation of compound 7 was synthesized (t-butyl sulfamide  N,N-dimethyl sulfamide) and found to have affinity Ki = 42 nM

7. Novel Structural Features of Newly Generated Ligands • Compound 8 exhibits a hydroxy-ketone backbone, a new functional group for HIV-1 protease inhibitors, which has been used in inhibitors of renin (a related aspartyl protease) • Compounds 10 & 11 have the backbones of initial structures 2 & 3 but have exchanged side chains, suggesting substituent transferability • Compound 12 is a “second-generation” result produced by taking initial structure 3 and replacing each end with groups from initial structure 1

8. Initial and Generated Compounds from Protein Kinase Inhibitors • New compound 24 produced from original structures 15 & 16 has been found to possess inhibitory activity against protein kinase p38 (IC50 = 160 nM) • Fewer compounds generated by BREED overall (abundance of rings limits the possible number of matching bonds)

9. Advantages of BREED • Efficiently automates recombination of molecule fragments to produce novel and effective inhibitors, avoiding tedious manual analysis • Relatively small number of initial structures required to generate large number of possibilities for compounds that may have enzyme inhibitory action • Use of experimentally determined ligands for initial structures eliminates need to search for docking conformation

10. Drawbacks to BREED • Structures of experimentally known compounds (ligands) still required for the process to get started • Few new structures generated for ring-rich inhibitors (ex. macrocycles/steroids) because of non-ring matching bond requirement • Some hybrids formed by conformational alterations that are missed by the current BREED algorithm (ex. the HIV-1 protease inhibitor below)

11. Sample Discussion Questions • (i) What changes could be made to the BREED algorithm to find ligands such as the protease inhibitor that given in example, without generating many inactive compounds? • (ii) What should be the major criteria for deciding which of the generated structures to first synthesize and test? Ex.: • Binding affinity • Novelty • “Druglikeness” • Solubility • Absorption into the circulatory system • Metabolic stability • Toxicity considerations