Gregor Bahrenberg

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.

Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice

The function of the transient receptor potential vanilloid 1 (TRPV1) cation channel was analyzed with RNA interference technologies and compared to TRPV1 knockout mice. Expression of shRNAs targeting TRPV1 in transgenic (tg) mice was proven by RNase protection assays, and TRPV1 downregulation was confirmed by reduced expression of TRPV1 mRNA and lack of receptor agonist binding in spinal cord membranes. Unexpectedly, TRPV3 mRNA expression was upregulated in shRNAtg but downregulated in knockout mice. Capsaicin-induced [Ca(2+)](i) changes in small diameter DRG neurons were significantly diminished in TRPV1 shRNAtg mice, and administration of capsaicin hardly induced hypothermia or nocifensive behaviour in vivo. Likewise, sensitivity towards noxious heat was reduced. Interestingly, spinal nerve injured TRPV1 knockout but not shRNAtg animals developed mechanical allodynia and hypersensitivity. The present study provides further evidence for the relevance of TRPV1 in neuropathic pain and characterizes RNA interference as valuable technique for drug target validation in pain research.

Molecular Determinants of Potent P2X2 Antagonism Identified by Functional Analysis, Mutagenesis, and Homology Docking

P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7′-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a “large urea” structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7′; and 3) the specific position of two sulfonic acid groups (3,3′; 6,6′) and of one methoxy group (1,1′) at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.

2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent transient receptor potential vanilloid 1 (TRPV1) antagonists: structure-activity relationships of 2-amino derivatives in the N-(6-trifluoromethylpyridin-3-ylmethyl) C-region.

A series of N-(2-amino-6-trifluoromethylpyridin-3-ylmethyl)-2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were designed combining previously identified pharmacophoric elements and evaluated as hTRPV1 antagonists. The SAR analysis indicated that specific hydrophobic interactions of the 2-amino substituents in the C-region of the ligand were critical for high hTRPV1 binding potency. In particular, compound 49S was an excellent TRPV1 antagonist (K(i(CAP)) = 0.2 nM; IC(50(pH)) = 6.3 nM) and was thus approximately 100- and 20-fold more potent, respectively, than the parent compounds 2 and 3 for capsaicin antagonism. Furthermore, it demonstrated strong analgesic activity in the rat neuropathic model superior to 2 with almost no side effects. Compound 49S antagonized capsaicin induced hypothermia in mice but showed TRPV1-related hyperthermia. The basis for the high potency of 49S compared to 2 is suggested by docking analysis with our hTRPV1 homology model in which the 4-methylpiperidinyl group in the C-region of 49S made additional hydrophobic interactions with the hydrophobic region.

2-(3-Fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists: structure activity relationships of the 2-oxy pyridine C-region.

The structure activity relationships of 2-oxy pyridine derivatives in the C-region of N-(6-trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as hTRPV1 antagonists were investigated. The analysis indicated that the lipophilicity of the 2-oxy substituents was critical for potent antagonism and 4 or 5 carbons appeared to be optimal for activity. Multiple compounds proved to have comparable activity to 1, which had been reported as the most potent antagonist for capsaicin activity among the previous series of compounds. Further analysis of compounds 22 (2-isobutyloxy) and 53 (2-benzyloxy) in the formalin test in mice demonstrated strong analgesic activity with full efficacy. Docking analysis of 53S using our hTRPV1 homology model indicated that the A- and B-region 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamide made important hydrophobic and hydrogen bonding interactions with Tyr511 and that the C-region 6-trifluoromethyl and 2-benzyloxy groups of pyridine occupied the two hydrophobic binding pockets, respectively.

Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor

P2X receptors are trimeric adenosine-5’-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.

2-Alkyl/alkenyl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-aryl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed highly potent TRPV1 antagonism toward capsaicin comparable to previous lead 7. Among them, compound 9 demonstrated anti-allodynia in a mouse neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of 9 with our hTRPV1 homology model provided insight into its specific binding mode.

TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.

α-Substituted 2-(3-fluoro-4-methylsulfonamidophenyl)acetamides as potent TRPV1 antagonists

A series of α-substituted acetamide derivatives of previously reported 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide leads (1, 2) were investigated for antagonism of hTRPV1 activation by capsaicin. Compound 34, which possesses an α-m-tolyl substituent, showed highly potent and selective antagonism of capsaicin with Ki(CAP)=0.1 nM. It thus reflected a 3-fold improvement in potency over parent 1. Docking analysis using our homology model indicated that the high potency of 34 might be attributed to a specific hydrophobic interaction of the m-tolyl group with the receptor.

Structure activity relationships of benzyl C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-substituted 4-(trifluoromethyl)benzyl C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The analysis indicated that the phenyl C-region derivatives exhibited better antagonism than those of the corresponding pyridine surrogates for most of the series examined. Among the phenyl C-region derivatives, the two best compounds 43 and 44S antagonized capsaicin selectively relative to their antagonism of other activators and showed excellent potencies with K(i(CAP))=0.3 nM. These two compounds blocked capsaicin-induced hypothermia, consistent with TRPV1 as their site of action, and they demonstrated promising analgesic activities in a neuropathic pain model without hyperthermia. The docking study of 44S in our hTRPV1 homology model indicated that its binding mode was similar with that of its pyridine surrogate in the A- and B-regions but displayed a flipped configuration in the C-region.