Improved Cav2.2 Channel Inhibitors through a gem-Dimethylsulfone Bioisostere Replacement of a Labile Sulfonamide.
ABSTRACT: We report the investigation of sulfonamide-derived Cav2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Cav2.2 potency without the liability of the circulating sulfonamide metabolite.
Project description:PI-103 (7) is a potent dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, but its rapid in vivo metabolism hinders its further clinical development. To improve the bioavailability of PI-103, we designed and synthesized a PI-103 bioisostere, PI-103BE (9) in which the phenolic hydroxyl group of PI-103 was replaced by a boronate, a structural modification known to enhance bioavailability of molecules containing phenolic hydroxyl moieties. In cell culture, PI-103BE is partially converted to its corresponding boronic acid (10) and to a lesser extent the active ingredient, PI-103. This mixture contributes to the in vitro activity of 9 that shows reduced potency compared to the parent compound. When administered to mice by oral gavage, 9 displays a significantly improved pharmacokinetic profile compared to PI-103, which shows no oral bioavailability at the same dose. Drug exposure of 9 as measured by the area under curve (AUC) value is 88.2 ng/mL*h for 7 and 8879.9 ng/mL*h for 10. When given by intraperitoneal injection (IP), the prodrug afforded an AUC of 32.3 ng/mL*h for 7 and 400.9 ng/mL*h for 10, compared to 9.7 ng/mL*h from PI-103 injection. In plasma from both pharmacokinetic studies, 9 is fully converted to 10 and 7, with the boronic acid metabolite (10) displaying antiproliferative activities comparable to 9, but weaker than 7. The boronic bioisostere of PI-103 thus offers an improved bioavailability that could be translated to in vivo efficacy of PI-103.
Project description:A chiral, hybrid bioisostere of the CF3 and Et groups (BITE) was installed in a series of vorinostat (Zolinza) analogues, and their histone deacetylase (HDAC) inhibitory behavior was studied relative to that of their nonfluorinated counterparts. Several of these compounds containing the 1,2-difluoroethylene unit showed in vitro potency greater than that of the clinically approved drug itself against HDAC1. This trend was found to be general with the BITE-modified HDAC inhibitors performing significantly better than the ethyl derivatives. Installed by the direct, catalytic vicinal difluorination of terminal alkenes using an I(I)/I(III) manifold, this underexplored chiral bioisostere shows potential in drug discovery.
Project description:BACKGROUND AND PURPOSE: ?-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs). EXPERIMENTAL APPROACH: Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively. KEY RESULTS: R10K substitution did not change the conserved ?-conotoxin backbone conformations of CVIE&F nor the ?-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ?-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ?-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID. CONCLUSIONS AND IMPLICATIONS: R10K substitution in CVIE&F significantly alters the kinetics of ?-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ?-conotoxins with optimized kinetic profiles for target binding.
Project description:Integration of voltage-gated Ca(2+) channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca(2+) channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the ?1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca(2+) channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba(2+) current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca(2+) channel activity in defined synaptic membrane compartments and thereby influences transmitter release.
Project description:N-type and P/Q-type calcium channels are documented players in the regulation of synaptic function; however, the mechanisms underlying their expression and cellular targeting are poorly understood. Ankyrin polypeptides are essential for normal integral membrane protein expression in a number of cell types, including neurons, cardiomyocytes, epithelia, secretory cells, and erythrocytes. Ankyrin dysfunction has been linked to defects in integral protein expression, abnormal cellular function, and disease. Here, we demonstrate that ankyrin-B associates with Cav2.1 and Cav2.2 in cortex, cerebellum, and brain stem. Additionally, using in vitro and in vivo techniques, we demonstrate that ankyrin-B, via its membrane-binding domain, associates with a highly conserved motif in the DII/III loop domain of Cav2.1 and Cav2.2. Further, we demonstrate that this domain is necessary for proper targeting of Cav2.1 and Cav2.2 in a heterologous system. Finally, we demonstrate that mutation of a single conserved tyrosine residue in the ankyrin-binding motif of both Cav2.1 (Y797E) and Cav2.2 (Y788E) results in loss of association with ankyrin-B in vitro and in vivo. Collectively, our findings identify an interaction between ankyrin-B and both Cav2.1 and Cav2.2 at the amino acid level that is necessary for proper Cav2.1 and Cav2.2 targeting in vivo.
Project description:We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse ?-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (G??-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.
Project description:Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 ?1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand-like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.
Project description:Alterations in dopamine receptor type 1 (D1R) density are associated with cognitive deficits of aging and schizophrenia. In the prefrontal cortex (PFC), D1R plays a critical role in the regulation of working memory, which is impaired in these cognitive deficit states, but the cellular events triggered by changes in D1R expression remain unknown. A previous report demonstrated that interaction between voltage-gated calcium channel type 2.2 (CaV2.2) and D1R stimulates CaV2.2 postsynaptic surface location in medial PFC pyramidal neurons. Here, we show that in addition to the occurrence of the physical receptor-channel interaction, constitutive D1R activity mediates up-regulation of functional CaV2.2 surface density. We performed patch-clamp experiments on transfected HEK293T cells and wild-type C57BL/6 mouse brain slices, as well as imaging experiments and cAMP measurements. We found that D1R coexpression led to ?60% increase in CaV2.2 currents in HEK293T cells. This effect was occluded by preincubation with a D1/D5R inverse agonist, chlorpromazine, and by replacing D1R with a D1R mutant lacking constitutive activity. Moreover, D1R-induced increase in CaV2.2 currents required basally active Gs protein, as well as D1R-CaV2.2 interaction. In mice, intraperitoneal administration of chlorpromazine reduced native CaV currents' sensitivity to ?-conotoxin-GVIA and their size by ?49% in layer V/VI pyramidal neurons from medial PFC, indicating a selective effect on CaV2.2. Additionally, we found that reducing D1/D5R constitutive activity correlates with a decrease in the agonist-induced D1/D5R inhibitory effect on native CaV currents. Our results could be interpreted as a stimulatory effect of D1R constitutive activity on the number of CaV2.2 channels available for dopamine-mediated modulation. Our results contribute to the understanding of the physiological role of D1R constitutive activity and may explain the noncanonical postsynaptic distribution of functional CaV2.2 in PFC neurons.
Project description:Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by 'fishing' for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3strep) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I-II loop or the distal half of the II-III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.
Project description:Voltage-gated calcium channels (VGCCs) are important mediators of pain hypersensitivity during inflammatory states, but their role in sensory nerve growth remains underexplored. Here, we assess the role of the N-type calcium channel Cav2.2 in the complete Freund's adjuvant (CFA) model of inflammatory pain. We demonstrate with <i>in situ</i> hybridization and immunoblotting, an increase in Cav2.2 expression after hind paw CFA injection in sensory neurons that respond to thermal stimuli, but not in two different mechanosensitive neuronal populations. Further, Cav2.2 upregulation post-CFA correlates with thermal but not mechanical hyperalgesia in behaving mice, and this hypersensitivity is blocked with a specific Cav2.2 inhibitor. Voltage clamp recordings reveal a significant increase in Cav2.2 currents post-CFA, while current clamp analyses demonstrate a significant increase in action potential frequency. Moreover, CFA-induced sensory nerve growth, which involves the extracellular signal-related kinase (ERK1/2) signaling pathway and likely contributes to inflammation-induced hyperalgesia, was blocked with the Cav2.2 inhibitor. Together, this work uncovers a role for Cav2.2 during inflammation, demonstrating that VGCC activity can promote thermal hyperalgesia through both changes in firing rates of sensory neurons as well as promotion of new neurite outgrowth.