Structure–activity relationships of simplified resiniferatoxin analogues with potent VR1 agonism elucidates an active conformation of RTX for VR1 binding Jeewoo Lee, a, * Su Yeon Kim, a Soyoung Park, a Ju-Ok Lim, a Ji-Min Kim, a Myungshim Kang, a Jiyoun Lee, a Sang-Uk Kang, a Hyun-Kyung Choi, a Mi-Kyung Jin, a Jacqueline D. Welter, b Tamas Szabo, b Richard Tran, b Larry V. Pearce, b Attila Toth b and Peter M. Blumberg b a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku, Seoul 151-742, South Korea b Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA Received 27 September 2003; accepted 9 December 2003 Abstract—We previously described a series of N-(3-acyloxy-2-benzylpropyl) homovanillate and N 0 -(4-hydroxy-3-methoxybenzyl) thiourea derivatives that were potent VR1 agonists with high-affinities and excellent analgesic profiles. The design of these simpli- fied RTX analogues was based on our RTX-derived pharmacophore model which incorporates the 4-hydroxy-3-methoxyphenyl (A- region), C 20 -ester (B-region), orthophenyl (C1-region) and C 3 -keto (C2-region) groups of RTX. For the purpose of optimizing the spatial arrangement of the four principal pharmacophores on the lead agonists (1–4), we have modified the distances in the parent C-region, 3-acyloxy-2-benzylpropyl groups, by lengthening or shortening one carbon to vary the distances between the pharmaco- phores. We find that two of the amides, 4 and 19, possess EC 50 values < 1 nM for induction of calcium influx in the VR1-CHO cells. As observed previously, the structure–activity relations for inhibition of RTX binding to VR1 and for induction of calcium uptake were distinct, presumably reflecting both intrinsic and methodological factors. In order to find the active conformation of VR1 ligands, the energy-minimized conformations of seven selected agonists were determined and the positions of their four pharmacophores were matched with those of five low energy RTX conformations. The rms values for the overlaps in the pharma- cophores were calculated and correlated with the measured binding affinities (K i ) and calcium influx (EC 50 ) values. The binding affinities of the agonists correlated best with the RMS values derived from RTX conformation E (r 2 =0.92), predicting a model of the active conformation of RTX and related vanilloids for binding to VR1. Poorer correlation was obtained between any of the conformations and the EC 50 values for calcium influx. # 2004 Elsevier Ltd. All rights reserved. 1. Introduction The vanilloid receptor 1 (VR1) 1 is a polymodal noci- ceptor activated by protons, 2 heat, 3 vanilloids such as capsaicin (CAP) 4 and resiniferatoxin (RTX), 5 and lipid mediators such as anandamide 6 and the lipid metabolic products of lipoxygenases. 7 VR1 has been cloned from rat, 3 human, 8 chicken 9 and guinea pig, 10 and has been shown to be a member of the vanilloid receptor (TRPV) subfamily of transient receptor potential (TRP) ion channels. 11 It is a cation channel with modest selectivity for calcium. Consistent with its complicated regulation, multiple sites of phosphorylation have been identi- fied 12,13 as well as multiple sites mediating acid respon- siveness, 14,15 and a site involved in inhibition by phosphoinositides. 16 Since VR1 is present pre- dominantly in nociceptors on primary sensory neuron, it has been an attractive therapeutic target for the treatment of pain and for other indications in which C- fiber sensory neurons are involved. Its validation as a target for analgesia has been confirmed recently by the observation that VR1 knockout mice exhibit deficits in the thermal hyperalgesia that accompanies tissue injury 0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2003.12.005 Bioorganic & Medicinal Chemistry 12 (2004) 1055–1069 Keywords: Vanilloid receptor 1; Resiniferatoxin. * Corresponding author. Tel.:+82-2-880-7846; fax:+82-2-888-0649; e-mail: jeewoo@snu.ac.kr