To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral linkers that derive from natural amino acid derivatives have been designed and synthesized. The new structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy synthesis and good inhibitory properties. Inhibitor (S)-9b exhibits a potency of 14.7 nM against nNOS and is 1134 and 322-fold more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 9b and propionate A on the heme and tetrahydrobiopterin (H4B) in nNOS, but not eNOS, contributes to its high selectivity. This work demonstrates the advantage of integrating known structures into structure optimization, and it should be possible to more readily develop compounds that incorporate bioavailability with these advanced features. Moreover, this integrative strategy is a general approach in new drug discovery.

Department of Biochemistry The University of Texas Health Science Center San Antonio TX 78384 7760 USA

Department of Biochemistry The University of Texas Health Science Center San Antonio TX 78384 7760 USA; Department of Pediatrics and Center for Applied Genomics 1st School of Medicine Charles University Prague Czech Republic

Department of Chemistry Northwestern University 2145 Sheridan Road Evanston IL 60208 3113 USA; Department of Molecular Biosciences Northwestern University 2145 Sheridan Road Evanston IL 60208 3113 USA; Chemistry of Life Processes Institute Northwestern University 2145 Sheridan Road Evanston IL 60208 3113 USA; Center for Molecular Innovation and Drug Discovery Northwestern University 2145 Sheridan Road Evanston IL 60208 3113 USA

Department of Molecular Biology and Biochemistry University of California Irvine CA 92697 USA; Department of Pharmaceutical Chemistry University of California Irvine CA 92697 USA; Department of Chemistry University of California Irvine CA 92697 USA

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