Discovery of Michael acceptor containing 1,4-dihydropyridines as first covalent inhibitors of L-/T-type calcium channels.

1,4-Dihydropyridines (DHPs) are an important class of blockers targeting different calcium channel subtypes and have great therapeutic value against cardiovascular and neurophysiologic conditions. Here, we present the design of DHP-based hexahydroquinoline derivatives as either selective or covalent inhibitors of calcium channels. These compounds were synthesized via a modified Hantzsch reaction under microwave irradiation and characterized by IR, 1H NMR, 13C NMR and mass spectra. Additionally, the proposed structure of HM12 was resolved by single crystal X-ray analysis. The abilities of the target compounds to block both L- and T-type calcium channels were evaluated by utilizing the whole-cell patch clamp technique. Our results identified covalent inhibitors of calcium channels for the first time, which could be achieved by introducing a Michael acceptor group into the ester side chain of the compounds. The proposed covalent binding between the compounds and the cysteine amino acid (Cys1492) within the DHP binding pocket of L-type calcium channel was supported by docking and pharmacophore analysis as well as a glutathione reactivity assay.

Synthesis of some new C2 substituted dihydropyrimidines and their electrophysiological evaluation as L-/T-type calcium channel blockers.

Drugs targeting different calcium channel subtypes have strong therapeutic potential for future drug development for cardiovascular disorders, neuropsychiatric diseases and cancer. This study aims to design and synthesize a new series of C2 substituted dihydropyrimidines to mimic the structure features of third generation long acting dihydropyridine calcium channel blockers and dihydropyrimidines analogues. The target compounds have been evaluated as blockers for CaV1.2 and CaV3.2 utilizing the whole-cell patch clamp technique. Among the tested compounds, compound 7a showed moderate calcium channel blockade activity against CaV3.2. Moreover, the predicted physicochemical properties and pharmacokinetic profiles of the target compounds recommend that they can be considered as drug-like candidates. The results highlight some significant information for the future design of lead compounds as calcium channel blockers.

TNF-α mediated upregulation of NaV1.7 currents in rat dorsal root ganglion neurons is independent of CRMP2 SUMOylation.

Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.

Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazoline derivatives for cytotoxic and analgesic effects.

As a bioisosteric strategy to overcome the poor metabolic stability of lead compound KYS05090S, a series of new fluoro-substituted 3,4-dihydroquinazoline derivatives was prepared and evaluated for T-type calcium channel (Cav3.2) block, cytotoxic effects and liver microsomal stability. Among them, compound 8h (KCP10068F) containing 4-fluorobenzyl amide and 4-cyclohexylphenyl ring potently blocked Cav3.2 currents (>90% inhibition) at 10µM concentration and exhibited cytotoxic effect (IC50=5.9µM) in A549 non-small cell lung cancer cells that was comparable to KYS05090S. Furthermore, 8h showed approximately a 2-fold increase in liver metabolic stability in rat and human species compared to KYS05090S. Based on these overall results, 8h (KCP10068F) may therefore represent a good backup compound for KYS05090S for further biological investigations as novel cytotoxic agent. In addition, compound 8g (KCP10067F) was found to partially protect from inflammatory pain via a blockade of Cav3.2 channels.

Activin C expressed in nociceptive afferent neurons is required for suppressing inflammatory pain.

Emerging evidence suggests that the suppressive modulators released from nociceptive afferent neurons contribute to pain regulation. However, the suppressive modulators expressed in small-diameter neurons of the dorsal root ganglion remain to be further identified. The present study shows that the activin C expressed in small dorsal root ganglion neurons is required for suppressing inflammation-induced nociceptive responses. The expression of activin C in small dorsal root ganglion neurons of rats was markedly downregulated during the early days of peripheral inflammation induced by intraplantar injection of the complete Freund's adjuvant. Intrathecal treatment with the small interfering RNA targeting activin ßC or the antibodies against activin C could enhance the formalin-induced nociceptive responses, and impair the recovery from the complete Freund's adjuvant-induced thermal hyperalgesia. Intrathecally applied activin C could reduce nociceptive responses induced by formalin or complete Freund's adjuvant. Moreover, activin C was found to inhibit the inflammation-induced phosphorylation of extracellular signal-regulated kinase in the dorsal root ganglia and the dorsal spinal cord. Thus, activin C functions as an endogenous suppressor of inflammatory nociceptive transmission and may have a therapeutic potential for treatment of inflammatory pain.

Follistatin-like 1 suppresses sensory afferent transmission by activating Na+,K+-ATPase.

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Inhibition of inflammatory pain by activating B-type natriuretic peptide signal pathway in nociceptive sensory neurons.

B-type natriuretic peptide (BNP) has been known to be secreted from cardiac myocytes and activate its receptor, natriuretic peptide receptor-A (NPR-A), to reduce ventricular fibrosis. However, the function of BNP/NPR-A pathway in the somatic sensory system has been unknown. In the present study, we report a novel function of BNP in pain modulation. Using microarray and immunoblot analyses, we found that BNP and NPR-A were expressed in the dorsal root ganglion (DRG) of rats and upregulated after intraplantar injection of complete Freund's adjuvant (CFA). Immunohistochemistry showed that BNP was expressed in calcitonin gene-related peptide (CGRP)-containing small neurons and IB4 (isolectin B4)-positive neurons, whereas NPR-A was present in CGRP-containing neurons. Application of BNP reduced the firing frequency of small DRG neurons in the presence of glutamate through opening large-conductance Ca2+-activated K+ channels (BKCa channels). Furthermore, intrathecal injection of BNP yielded inhibitory effects on formalin-induced flinching behavior and CFA-induced thermal hyperalgesia in rats. Blockade of BNP signaling by BNP antibodies or cGMP-dependent protein kinase (PKG) inhibitor KT5823 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester] impaired the recovery from CFA-induced thermal hyperalgesia. Thus, BNP negatively regulates nociceptive transmission through presynaptic receptor NPR-A, and activation of the BNP/NPR-A/PKG/BKCa channel pathway in nociceptive afferent neurons could be a potential strategy for inflammatory pain therapy.

Inflammation and nerve injury induce expression of pancreatitis-associated protein-II in primary sensory neurons.

Pancreatitis-associated protein (PAP)-I and -II, lectin-related secretory proteins, are members of the regenerating gene (Reg) family. Although expression of PAP-I was found in the dorsal root ganglion (DRG) neurons following peripheral nerve injury and cystitis, whether PAP-II could be expressed in DRG neurons in chronic pain models remains unclear. The present study shows an inflammation- and nerve injury-triggered expression of PAP-II in rat DRG neurons. In situ hybridization showed that only a few DRG neurons normally contained PAP-I and -II mRNAs. After peripheral inflammation, PAP-I and -II mRNAs were present in over half of small DRG neurons. Such an elevated expression of PAP-I and -II reached the peak level on the second day. Immunostaining showed that the expression of PAP-II was mostly increased in the isolectin B4-positive subset of small DRG neurons after inflammation. Furthermore, the expression of PAP-II was also induced in DRG neurons after peripheral nerve injury. Interestingly, PAP-II expression was shifted from small neurons on day 2 to large DRG neurons that expressed neuropeptide Y during the later post-injury days. These results suggest that PAP-II may play potential roles in the modulation of spinal sensory pathways in pathological pain states.

Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion.

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.