1 Synthesis and Evaluation of Bifunctional sGC Regulators: 2 Optimization of a Connecting Linker 3 Mikolaj Chromiń ski, † Lukasz Banach, † Maksymilian Karczewski, † Keith ó Proinsias, † Iraida Sharina, ‡ 4 Dorota Gryko,* ,† and Emil Martin* ,‡ 5 † Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland 6 ‡ Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center in Houston, The University of 7 Texas, 1941 East Road, Houston, Texas 77054, United States 8 * S Supporting Information 9 ABSTRACT: Hybrid molecules composed of PpIX and cobyrinic acid derivatives conjugated through 10 linkers of varying length and composition were prepared via 1,3-dipolar cycloaddition (CuAAC) or 11 amidation/esteryfication reactions. They were tested for activation of soluble guanylyl cyclase (sGC), a 12 key enzyme in the NO/cGMP signaling pathway, by an in vitro GTP→cGMP conversion assay. Using 13 purified heme-deficient sGC and truncated sGC variants lacking a heme-binding domain, we 14 demonstrate that such hybrid molecules may activate sGC by targeting heme-binding and/or catalytic 15 domain. While all conjugates activated sGC, only selected compounds served as bifunctional regulators 16 and were capable of simultaneous targeting both heme and catalytic domains of sGC. The length and 17 type of a linker connecting both components had a profound effect on the extent of sGC activation, 18 indicating that the linker’s type is crucial for their binding affinities with regulatory and catalytic 19 domains. Only hybrids with the conjugated linker of 13-16 atom length synergistically target both 20 domains and displayed the lowest EC 50 and highest activating potency. Compounds with shorter 21 connecting linkers were much less potent and were no more active than the cobyrinic acid component alone The most active 22 conjugate, which showed a 60-fold activation of sGC, was compound 11, in which PpIX and cobyrinic acid components are 23 separated by 11 atoms chain with the triazole moiety in between. 24 ■ INTRODUCTION 25 Soluble guanylyl cyclase (sGC) is the principal intracellular 26 receptor for nitric oxide (NO). In response to NO binding to 27 the sGC heme group, the conversion of guanosine triphosphate 28 (GTP) into cyclic guanosine-3′,5′monophosphate (cGMP) is 29 enhanced several hundred fold. sGC is a heterodimeric protein 30 composed of α- and β-subunits. 1 Although two isoforms for 31 each subunit have been identified (α1, α2 and β1, β2), only 32 heterodimers containing the β1 subunit are catalytically active 33 and responsive to the NO stimulus. 2 The α1β1 heterodimer is 34 the predominant sGC enzyme with almost ubiquitously 35 expression. While the X-ray structure of full-length sGC is 36 not yet available, structure-activity studies clearly identify three 37 independent domains: regulatory, catalytic, and dimerization 38 region, with specific functions integrated into the heterodimer. 3 39 C-Terminal regions of each subunits form the catalytic domain f1 40 and both subunits are essential for cGMP synthesis (Figure 1, 41 CAT domains). Furthermore, the interaction between central 42 domains of α- and β-subunits contributes to the formation of a 43 stable heterodimer (CC and PAS domains) and mediation of 44 stimulatory signal induced in the N-terminal regulatory domain 45 (PAS and HNOX domains). N-Terminal regions of both 46 subunits are critical for sGC activation because the N-terminal 47 part of the β subunit harbors the heme moiety (βHNOX), 48 while the N-terminal α subunit is involved in the interaction 49 with allosteric stimulators of sGC (αHNOX). 50 Under proper physiological conditions, binding of NO to the 51 heme moiety induces a set of transformations leading to 52 enhanced cGMP synthesis. Thus, activated sGC is a key 53 component in the NO/cGMP signaling that governs various 54 physiological processes. These include, but are not limited to, 55 vascular smooth muscle relaxation, electrolyte homeostasis, 56 platelet function, neurotransmission, mitochondrial neogenesis, 57 etc. 6 Many pathological conditions lead to impaired bio- Received: May 14, 2013 Figure 1. Schematic representation of sGC architecture. Shown is the hypothetical orientation of domains in α1β1 sGC heterodimer based on previous studies. 4,5 αCAT and βCAT, guanylyl cyclase catalytic domains; αCC and βCC, coil-coil elements; αPAS and βPAS, PAS- like regions; βHNOX, heme-NO/oxygen binding domain of the β subunit; αHNOX, amino-terminal domain of the α subunit. Article pubs.acs.org/jmc © XXXX American Chemical Society A dx.doi.org/10.1021/jm400715h | J. Med. Chem. XXXX, XXX, XXX-XXX cdz00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.5.i3 HF03:4109 | 2.0 alpha 39) 2013/07/22 11:27:00 | PROD-JCAVA | rq_2698947 | 8/28/2013 15:54:33 | 18 | JCA-DEFAULT