GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

A novel dual mode-of-action anti-hyperalgesic compound in rats which is neuroprotective and promotes neuroregeneration.

Chronic neuropathic pain (CNP) can result from surgery or traumatic injury, but also from peripheral neuropathies caused by diseases, viral infections, or toxic treatments. Opioids, although very effective for acute pain, do not prevent the development of CNP, and are considered as insufficient treatment. Therefore, there is high need for effective and safe non-opioid options to treat, prevent and eventually reverse CNP. A more effective approach to alleviating CNP would constitute a treatment that acts concurrently on various mechanisms involved in relieving pain symptoms and preventing or reversing chronification by enhancing both neuroprotection and neuroregeneration. We have identified and characterized GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-caboxylic acid amide), a novel drug which is able to activate both voltage-gated potassium channels of the Kv7 family and the mitochondrial translocator protein 18 kDa (TSPO). The dual mode-of-action (MoA) of GRT-X was indicated in in vitro studies and in vivo in a rat model of diabetic neuropathy. In this model, mechanical hyperalgesia was dose-dependently inhibited. After severe crush lesion of cervical spinal nerves in rats, GRT-X promoted survival, speeded up regrowth of sensory and motor neurons, and accelerated recovery of behavioral and neuronal responses to heat, cold, mechanical and electrical stimuli. These properties may reduce the likelihood of chronification of acute pain, and even potentially relieve established CNP. The absence of a conditioned place preference in rats suggests lack of abuse potential. In conclusion, GRT-X offers a promising preclinical profile with a novel dual MoA.

2-(Halogenated Phenyl) acetamides and propanamides as potent TRPV1 antagonists.

A series consisting of 117 2-(halogenated phenyl) acetamide and propanamide analogs were investigated as TRPV1 antagonists. The structure-activity analysis targeting their three pharmacophoric regions indicated that halogenated phenyl A-region analogs exhibited a broad functional profile ranging from agonism to antagonism. Among the compounds, antagonists 28 and 92 exhibited potent antagonism toward capsaicin for hTRPV1 with Ki[CAP] = 2.6 and 6.9 nM, respectively. Further, antagonist 92 displayed promising analgesic activity in vivo in both phases of the formalin mouse pain model. A molecular modeling study of 92 indicated that the two fluoro groups in the A-region made hydrophobic interactions with the receptor.

Discovery of 1-(1H-indazol-4-yl)-3-((1-phenyl-1H-pyrazol-5-yl)methyl) ureas as potent and thermoneutral TRPV1 antagonists.

A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure-activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4-0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.

RNA interference-mediated silencing of Kv7.2 in rat dorsal root ganglion neurons abolishes the anti-nociceptive effect of a selective channel opener.

INTRODUCTION: Development of agonistic analgesic drugs requires proof of selectivity in vivo attainable by selective antagonists or several knockdown strategies. The Kv7.2 potassium channel encoded by the KCNQ2 gene regulates neuronal excitability and its activation inhibits nociceptive transmission. Although it is a potentially attractive target for analgesics, no clinically approved Kv7.2 agonists are currently available and selectivity of drug candidates is hard to demonstrate in vivo due to the expenditure to generate KCNQ2 knockout animals and the lack of Kv7.2 selective antagonists. The present study describes the set-up of an RNA interference-based model that allows studying the selectivity of Kv7.2 openers. METHODS: Adeno-associated virus (AAV) vectors were used to deliver the expression cassette for a short hairpin RNA targeting KCNQ2. Heat nociception was tested in rats after intrathecal AAV treatment. RESULTS: Surprisingly, screening of AAV serotypes revealed serotype 7, which has rarely been explored, to be best suited for transduction of dorsal root ganglia neurons following intrathecal injection. Knockdown of the target gene was confirmed by qRT-PCR and the anti-nociceptive effect of a Kv7.2 agonist was found to be completely abolished by the treatment. DISCUSSION: We consider this approach not only to be suitable to study the selectivity of novel analgesic drugs targeting Kv7.2, but rather to serve as a general fast and simple method to generate functional and phenotypic knockdown animals during drug discovery for central and peripheral pain targets.

In vitro characterization of the thermoneutral transient receptor potential vanilloid-1 (TRPV1) inhibitor GRTE16523.

The TRPV1 ion channel is a neuronal sensor that plays an important role in nociception and neuropathic as well as inflammatory pain. In clinical trials, hyperthermia and thermo-hypoaesthesia turned out as major side effects of TRPV1 antagonists, preventing successful development of such molecules as analgesics. In vitro studies demonstrated that the TRPV1 ion channel is a polymodal sensor integrating stimuli from molecular modulators with temperature, pH and transmembrane potential. Temperature dependent gating is suggested to constitute the molecular basis for its role in heat sensation and body temperature regulation. Drug discovery scientists since many years seek to obtain "thermoneutral" TRPV1 inhibitors, blocking the channels sensitivity for painful stimuli while keeping its temperature mode of activation unaffected. Aiming for a screening rational for the identification of thermoneutral TRPV1 antagonists, we broadly characterized the prototypic small molecule TRPV1 inhibitors GRT12360V and GRTE16523. In vitro, GRT12360V demonstrated pan-modality inhibition on human, cynomolgus and rodent TRPV1, whereas GRTE16523 selectively bypassed the channels temperature mode on human and cynomolgus TRPV1 and revealed partial agonism on rodent channels. Strikingly, in vivo, GRT12360V induced hyperthermia in all species tested whereas GRTE16523 proved thermoneutral in cynomolgus monkeys and induced hypothermia in rodents. Hence, working out the different in vitro to in vivo correlations of two compounds, we suggest temperature dependent voltage gating as key parameter when screening for thermoneutral TRPV1 inhibitors. We highlight a species difference of molecular TRPV1 pharmacology between primates and rodents and provide a methodological breakthrough to engineer thermoneutral TRPV1 antagonists with improved therapeutic safety.

Discovery of indane propanamides as potent and selective TRPV1 antagonists.

A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.

Fragment-Based Discovery of Novel Potent Sepiapterin Reductase Inhibitors.

Genome-wide-association studies in chronic low back pain patients identified sepiapterin reductase as a high interest target for developing new analgesics. Here we used 19F NMR fragment screening for the discovery of novel, ligand-efficient SPR inhibitors. We report the crystal structures of six chemically diverse inhibitors complexed with SPR, identifying relevant interactions and binding modes in the sepiapterin pocket. Exploration of our initial fragment screening hit led to double-digit nanomolar inhibitors of SPR with excellent ligand efficiency.

Inhibiting the immunoproteasome's ß5i catalytic activity affects human peripheral blood-derived immune cell viability.

Small molecule inhibitors selectively targeting the immunoproteasome subunit ß5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss-of-function mutations in the gene encoding ß5i (Psmb8) suffer from the proteasome-associated autoinflammatory syndromes (PRAAS) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective ß5i inhibitor ONX 0914 and Bortezomib, a pan-proteasome inhibitor, in human peripheral blood mononuclear cells (PBMCs). Both compounds efficiently blocked pro-inflammatory cytokine secretion in human whole blood and PBMC cultures stimulated with toll-like receptor (TLR) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen CMVpp65 or by polyclonal stimulation. The viability of PBMCs, however, was rapidly decreased in the presence of ONX 0914 and Bortezomib demonstrated by decreased residual cytosolic ATP and increased Annexin V surface binding. Interestingly, HLA-DR + monocytes were rapidly depleted from the cultures in the presence of ONX 0914 as a ß5i-selective inhibitor and Bortezomib. In conclusion, the anti-inflammatory potential of ß5i-selective inhibitors is correlating with a cytotoxicity increase in human PBMC subsets ex vivo. Our results provide important insights into the anti-inflammatory mechanism of action of ß5i-inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.

4-Aminophenyl acetamides and propanamides as potent transient receptor potential vanilloid 1 (TRPV1) ligands.

A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.

Discovery of 2-(3,5-difluoro-4-methylsulfonaminophenyl)propanamides as potent TRPV1 antagonists.

A series of A-region analogues of 2-(3-fluoro-4-methylsufonamidophenyl) propanamide 1 were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that a fluoro group at the 3- (or/and) 5-position and a methylsulfonamido group at the 4-position were optimal for antagonism of TRPV1 activation by capsaicin. The most potent antagonist 6 not only exhibited potent antagonism of activation of hTRPV1 by capsaicin, low pH and elevated temperature but also displayed highly potent antagonism of activation of rTRPV1 by capsaicin. Further studies demonstrated that antagonist 6 blocked the hypothermic effect of capsaicin in vivo, consistent with its in vitro mechanism, and it showed promising analgesic activity in the formalin animal model.

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists.

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with Ki(CAP)=0.1nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.

t-Butyl pyridine and phenyl C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-substituted 6-t-butylpyridine and 4-t-butylphenyl C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The analysis of structure activity relationships indicated that the pyridine derivatives generally exhibited a little better antagonism than did the corresponding phenyl surrogates for most of the series. Among the compounds, compound 7 showed excellent antagonism toward capsaicin activation with Ki=0.1nM and compound 60S demonstrated a strong antiallodynic effect with 83% MPE at 10mg/kg in the neuropathic pain model. The docking study of 7S in our hTRPV1 homology model indicated that the interactions between the A/B-regions of 7S with Tyr511 and the interactions between the t-butyl and ethyl groups in the C-region of 7S with the two hydrophobic binding pockets of hTRPV1 contributed to the high potency.

Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614.

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.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-ß1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

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.

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

A series of pyridine derivatives in the C-region of N-((6-trifluoromethyl-pyridin-3-yl)methyl) 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. The SAR analysis indicated that 6-difluorochloromethyl pyridine derivatives were the best surrogates of the C-region for previous leads. Among them, compound 31 showed excellent antagonism to capsaicin as well as to multiple hTRPV1 activators. It demonstrated strong analgesic activity in the formalin test in mice with full efficacy and it blocked capsaicin-induced hypothermia in vivo.