Discovery of N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea as a potent TRPV1 antagonistic template.

A series of homologous analogues of prototype antagonist 1 and its urea surrogate were investigated as hTRPV1 ligands. Through one-carbon elongation in the respective pharmacophoric regions, N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea was identified as a novel and potent TRPV1 antagonistic template. Its representative compound 27 showed a potency comparable to that of lead compound 1. Docking analysis of compound 27 in our hTRPV1 homology model indicated that its binding mode was similar with that of 1S.

2-Sulfonamidopyridine C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-sulfonamidopyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide were investigated as hTRPV1 ligands. Systematic modification on the 2-sulfonamido group provided highly potent TRPV1 antagonists. The N-benzyl phenylsulfonamide derivatives 12 and 23 in particular showed higher affinities than that of lead compound 1. Compound 12 exhibited strong analgesic activity in the formalin pain model. Docking analysis of its chiral S-form 12S in our hTRPV1 homology model indicated that its high affinity might arise from additional hydrophobic interactions not present in lead compound 1S.

Structural insight into tetrameric hTRPV1 from homology modeling, molecular docking, molecular dynamics simulation, virtual screening, and bioassay validations.

The transient receptor potential vanilloid type 1 (TRPV1) is a heat-activated cation channel protein, which contributes to inflammation, acute and persistent pain. Antagonists of human TRPV1 (hTRPV1) represent a novel therapeutic approach for the treatment of pain. Developing various antagonists of hTRPV1, however, has been hindered by the unavailability of a 3D structure of hTRPV1. Recently, the 3D structures of rat TRPV1 (rTRPV1) in the presence and absence of ligand have been reported as determined by cryo-EM. rTRPV1 shares 85.7% sequence identity with hTRPV1. In the present work, we constructed and reported the 3D homology tetramer model of hTRPV1 based on the cryo-EM structures of rTRPV1. Molecular dynamics (MD) simulations, energy minimizations, and prescreen were applied to select and validate the best model of hTRPV1. The predicted binding pocket of hTRPV1 consists of two adjacent monomers subunits, which were congruent with the experimental rTRPV1 data and the cyro-EM structures of rTRPV1. The detailed interactions between hTRPV1 and its antagonists or agonists were characterized by molecular docking, which helped us to identify the important residues. Conformational changes of hTRPV1 upon antagonist/agonist binding were also explored by MD simulation. The different movements of compounds led to the different conformational changes of monomers in hTRPV1, indicating that TRPV1 works in a concerted way, resembling some other channel proteins such as aquaporins. We observed that the selective filter was open when hTRPV1 bound with an agonist during MD simulation. For the lower gate of hTRPV1, we observed large similarities between hTRPV1 bound with antagonist and with agonist. A five-point pharmacophore model based on several antagonists was established, and the structural model was used to screen in silico for new antagonists for hTRPV1. By using the 3D TRPV1 structural model above, the pilot in silico screening has begun to yield promising hits with activity as hTRPV1 antagonists, several of which showed substantial potency.

Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands.

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.

α-Methylated simplified resiniferatoxin (sRTX) thiourea analogues as potent and stereospecific TRPV1 antagonists.

A series of α-methylated analogues of the potent sRTX thiourea antagonists were investigated as rTRPV1 ligands in order to examine the effect of α-methylation on receptor activity. The SAR analysis indicated that activity was stereospecific with the (R)-configuration of the newly formed chiral center providing high binding affinity and potent antagonism while the configuration of the C-region was not significant.

Structure-activity relationships and molecular modeling of the N-(3-pivaloyloxy-2-benzylpropyl)-N'-[4-(methylsulfonylamino)benzyl] thiourea template for TRPV1 antagonism.

The structure-activity relationships of N-(3-acyloxy-2-benzylpropyl)-N'-4-[(methylsulfonylamino)benzyl] thioureas, which represent simplified RTX-based vanilloids, were investigated by varying the distances between the four principal pharmacophores and assessing binding and antagonistic activity on rTRPV1. The analysis indicated that a 3-pivaloyloxy-2-benzylpropyl C-region conferred the best potency in binding affinity and antagonism. The molecular modeling of this best template with the tetrameric homology model of rTRPV1 was performed to identify its binding interactions with the receptor.

The SAR analysis of TRPV1 agonists with the α-methylated B-region.

A series of TRPV1 agonists with amide, reverse amide, and thiourea groups in the B-region and their corresponding α-methylated analogues were investigated. Whereas the α-methylation of the amide B-region enhanced the binding affinities and potencies as agonists, that of the reverse amide and thiourea led to a reduction in receptor affinity. The analysis indicated that proper hydrogen bonding as well as steric effects in the B-region are critical for receptor binding.

N-4-t-Butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the A-region.

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.

2-(4-Methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the B and C-regions.

On the basis of the previous lead N-4-t-butylbenzyl 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamide (3) as a potent TRPV1 antagonist, structure-activity relationships for the B (propanamide part) and C-region (4-t-butylbenzyl part) have been investigated for rTRPV1 in CHO cells. The B-region was modified with dimethyl, cyclopropyl and reverse amides and then the C-region was replaced with 4-substituted phenyl, aryl alkyl and diaryl alkyl derivatives. Among them, compound 50 showed high binding affinity with K(i)=21.5nM, which was twofold more potent than 3 and compound 54 exhibited potent antagonism with K(i(ant))=8.0nM comparable to 3.

Receptor activity and conformational analysis of 5'-halogenated resiniferatoxin analogs as TRPV1 ligands.

A series of 5'-halogenated resiniferatoxin analogs have been investigated in order to examine the effect of halogenation in the A-region on their binding and the functional pattern of agonism/antagonism for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Halogenation at the 5-position in the A-region of RTX and of 4-amino RTX shifted the agonism of parent compounds toward antagonism. The extent of antagonism was greater as the size of the halogen increased (I > Br > Cl > F) while the binding affinities were similar, as previously observed for our potent agonists. In this series, 5-bromo-4-amino RTX (39) showed very potent antagonism with K(i) (ant) = 2.81 nM, which was thus 4.5-fold more potent than 5'-iodo RTX, previously reported as a potent TRPV1 antagonist. Molecular modeling analyses with selected agonists and the corresponding halogenated antagonists revealed a striking conformational difference. The 3-methoxy of the A-region in the agonists remained free to interact with the receptor whereas in the case of the antagonists, the compounds assumed a bent conformation, permitting the 3-methoxy to instead form an internal hydrogen bond with the C4-hydroxyl of the diterpene.

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies.

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1-TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic DAG-Lactones as PKC Activators.

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

Discovery of Benzopyridone-Based Transient Receptor Potential Vanilloid 1 Agonists and Antagonists and the Structural Elucidation of Their Activity Shift.

Among a series of benzopyridone-based scaffolds investigated as human transient receptor potential vanilloid 1 (TRPV1) ligands, two isomeric benzopyridone scaffolds demonstrated a consistent and distinctive functional profile in which 2-oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoquinolin-5-yl analogues (e.g., 3) showed full agonism with high potency. Our computational models provide insight into the agonist-antagonist boundary of the analogues suggesting that the Arg557 residue in the S4-S5 linker might be important for sensing the agonist binding and transmitting signals. These results provide structural insights into the TRPV1 and the protein-ligand interactions at a molecular level.

Polar 3-alkylidene-5-pivaloyloxymethyl-5'-hydroxymethyl-gamma-lactones as protein kinase C ligands and antitumor agents.

A series of DAG-lactones with polar 3-alkylidene substituents have been investigated as PKC-alpha ligands and antitumor agents. Extensive analysis of structure-activity relationships for the 3-alkylidene chain revealed that polar groups such as ether, hydroxyl, aldehyde, ester, acyloxy, and amido were tolerated with similar binding affinities and reduced lipophilicities compared to the corresponding unsubstituted alkylidene chain. Among the derivatives, compounds 5, 6 and 8 with an ether type of side chain showed high binding affinities in range of K(i)= 3-5 nM and excellent antitumor profiles, particularly against the colo205 colon cancer and the K562 leukemia cell lines.

Halogenation of 4-hydroxy/amino-3-methoxyphenyl acetamide TRPV1 agonists showed enhanced antagonism to capsaicin.

As an extension of our analysis of the effect of halogenation on thiourea TRPV1 agonists, we have now modified selected 4-hydroxy(or 4-amino)-3-methoxyphenyl acetamide TRPV1 agonists by 5- or 6-halogenation on the aromatic A-region and evaluated them for potency for TRPV1 binding and regulation and for their pattern of agonism/antagonism (efficacy). Halogenation shifted the functional activity at TRPV1 toward antagonism with a greater extent of antagonism as the size of the halogen increased (I>Br>Cl), as previously observed for the thiourea series. The extent of antagonism was greater for halogenation at the 5-position than at the 6-position, in contrast to SAR for the thiourea series. In this series, compounds 55 and 75 showed the most potent antagonism, with K(i) (ant)=2.77 and 2.19nM, respectively, on rTRPV1 expressed in Chinese hamster ovary cells. The compounds were thus ca. 40-60-fold more potent than 6'-iodononivamide.

Discovery of Nonpungent Transient Receptor Potential Vanilloid 1 (TRPV1) Agonist as Strong Topical Analgesic.

Paradoxically, some TRPV1 agonists are, at the organismal level, both nonpungent and clinically useful as topical analgesics. Here, we describe the scaled-up synthesis and characterization in mouse models of a novel, nonpungent vanilloid. Potent analgesic activity was observed in models of neuropathic pain, and the compound blocked capsaicin induced allodynia, showing dermal accumulation with little transdermal absorption. Finally, it displayed much weaker systemic toxicity compared to capsaicin and was negative in assays of genotoxicity.

Non-vanillyl resiniferatoxin analogues as potent and metabolically stable transient receptor potential vanilloid 1 agonists.

A series of non-vanillyl resiniferatoxin analogues, having 4-methylsulfonylaminophenyl and fluorophenyl moieties as vanillyl surrogates, have been investigated as ligands for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Although lacking the metabolically problematic 4-hydroxy substituent on the A-region phenyl ring, the compounds retained substantial agonist potency. Indeed, the 3-methoxy-4-methylsulfonylaminophenyl analog (1) was modestly (2.5-fold) more potent than RTX, with an EC(50)=0.106 nM. Further, it resembled RTX in its kinetics and pattern of stimulation of the levels of intracellular calcium in individual cells, as revealed by imaging. Compound 1 displayed modestly enhanced in vitro stability in rat liver microsomes and in plasma, suggesting that it might be a pharmacokinetically more favorable surrogate of resiniferatoxin. Molecular modeling analyses with selected analogues provide evidence that the conformational differences could affect their binding affinities, especially for the ester versus amide at the B-region.

Combination of a Rapidly Penetrating Agonist and a Slowly Penetrating Antagonist Affords Agonist Action of Limited Duration at the Cellular Level.

The capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) has been an object of intense interest for pharmacological development on account of its critical role in nociception. In the course of structure activity analysis, it has become apparent that TRPV1 ligands may vary dramatically in the rates at which they interact with TRPV1, presumably reflecting differences in their abilities to penetrate into the cell. Using a fast penetrating agonist together with an excess of a slower penetrating antagonist, we find that we can induce an agonist response of limited duration and, moreover, the duration of the agonist response remains largely independent of the absolute dose of agonist, as long as the ratio of antagonist to agonist is held constant. This general approach for limiting agonist duration under conditions in which absolute agonist dose is variable should have more general applicability.

Discovery of dual-acting opioid ligand and TRPV1 antagonists as novel therapeutic agents for pain.

In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting. Based on a combination approach, a series of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for their receptor activities. Among them, compound 49 exhibited the most promising dual-acting activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo,49 displayed potent, dose-dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting TRPV1 antagonist/mu-opioid ligands can be made and represent a new and promising class of analgesic.

Stereospecific high-affinity TRPV1 antagonists: chiral N-(2-benzyl-3-pivaloyloxypropyl) 2-[4-(methylsulfonylamino)phenyl]propionamide analogues.

Previously, we reported the thiourea antagonists 2a and 2b as potent and high affinity TRPV1 antagonists. For further optimization of the lead compounds, a series of their amide and alpha-substituted amide surrogates were investigated and novel chiral N-(2-benzyl-3-pivaloyloxypropyl) 2-[4-(methylsulfonylamino)phenyl]propionamide analogues were characterized as potent and stereospecific rTRPV1 antagonists. In particular, compounds 72 and 73 displayed high binding affinities, with K i values of 4.12 and 1.83 nM and potent antagonism with K i values of 0.58 and 5.2 nM, respectively, in rTRPV1/CHO cells. These values are comparable or more potent than those of 5-iodoRTX under the same assay conditions. A distinctive binding model that includes two hydrophobic pockets is proposed for this series of compounds based on docking studies of 57 and 72 with a homology model of the TM3/4 region of TRPV1.