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Due to the central role of tubulin in various cellular functions, it is a validated target for anti-cancer therapeutics. However, many of the current tubulin inhibitors are derived ...from complex natural products and suffer from multidrug resistance, low solubility, toxicity issues, and/or the lack of multi-cancer efficacy. As such, there is a continued need for the discovery and development of new anti-tubulin drugs to enter the pipeline. Herein we report on a group of indole-substituted furanones that were prepared and tested for anti-cancer activity. Molecular docking studies showed positive correlations between favorable binding in the colchicine binding site (CBS) of tubulin and anti-proliferative activity, and the most potent compound was found to inhibit tubulin polymerization. These compounds represent a promising new structural motif in the search for small heterocyclic CBS cancer inhibitors.
Our homology molecular model of the open/inactivated state of the Na + channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below
the ...selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's
positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level
on permeation in hNa V 1.5 we replaced Phe-1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively
charged residues as well as the neutral cysteine and alanine. These mutations eliminated use-dependent lidocaine block with
no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel
conductance (γ) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased
γ by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations
affected Na + /K + selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest
positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate
both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted
changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic
field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge.
Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face ...the inner pore just below the selectivity filter, is critical in use-dependent drug block.
Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes.
The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed.
These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.
Mibefradil is a T-type Ca2+ channel antagonist with reported cross-reactivity with other classes of ion channels, including K+, Cl-, and Na+ channels. Using whole-cell voltage clamp, we examined ...mibefradil block of four Na+ channel isoforms expressed in human embryonic kidney cells: Nav1.5 (cardiac), Nav1.4 (skeletal muscle), Nav1.2 (brain), and Nav1.7 (peripheral nerve). Mibefradil blocked Nav1.5 in a use/frequency-dependent manner, indicating preferential binding to states visited during depolarization. Mibefradil blocked currents of all Na+ channel isoforms with similar affinity and a dependence on holding potential, and drug off-rate was slowed at depolarized potentials (k(off) was 0.024/s at -130 mV and 0.007/s at -100 mV for Nav1.5). We further probed the interaction of mibefradil with inactivated Nav1.5 channels. Neither the degree nor the time course of block was dependent on the stimulus duration, which dramatically changed the residency time of channels in the fast-inactivated state. In addition, inhibiting the binding of the fast inactivation lid (Nav1.5 ICM + MTSET) did not alter mibefradil block, confirming that the drug does not preferentially interact with the fast-inactivated state. We also tested whether mibefradil interacted with slow-inactivated state(s). When selectively applied to channels after inducing slow inactivation with a 60-s pulse to -10 mV, mibefradil (1 microM) produced 45% fractional block in Nav1.5 and greater block (88%) in an isoform (Nav1.4) that slow-inactivates more completely. Our results suggest that mibefradil blocks Na+ channels in a state-dependent manner that does not depend on fast inactivation but probably involves interaction with one or more slow-inactivated state(s).
We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation ...include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development.
The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein
kinase in vitro and block proinflammatory cytokine production in vivo ...has been determined. Using Edman sequencing, 125 I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli -expressed p38 kinase at Thr 175 , which is proximal to the ATP binding site. Titration calorimetric studies with E. coli -expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation
of p38 kinase with the ATP analogue, FSBA (5â²- p -(fluorosulfonyl)benzoyladenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the
nonphosphorylated enzyme was inhibited by SB203580 with a K
m of 9.6 m m . Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was
competitive with ATP with a K
i of 21 n m and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125 I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl
imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences
in nonconserved regions within or near the ATP binding pocket.
Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and ...radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.
The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38 ) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 ...insect cells.
Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were
identified as resulting from cleavages after Glu 19 , Ser 98 , and Glu 110 . The mature enzyme was composed of mixture of enzymes with N termini of Gly 113 , Arg 114 , and Ala 115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate
in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide
linkage, and that the protein had Met 329 as the C-terminal residue.
A mutant was constructed in which the active site Cys 139 was changed to Ser. Ser 139 ,Ala 163 Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the
presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the
activation of wild-type. Ser 139 ,Ala 163 Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation
mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.
The role of the outermost three charged residues of Domain IV/S4 in controlling gating of Ca(v)3.2 was investigated using single substitutions of each arginine with glutamine, cysteine, histidine, ...and lysine in a Flp-In-293 cell line, in which expression levels could be compared. Channel density, based on gating charge measurements, was ~125,000 channels/cell (10 fC/pF), except for R2Q and R3C, which expressed at lower levels. Channels substituted at Arg-1715 (R1C, R1Q, R1H) demonstrated such modest changes that a role in voltage sensing could not be determined. Arg-1718 (R2) made a contribution to activation voltage sensing, and the channel was sensitive to the geometry of side-chain substitutions at this position. Arg-1721 (R3) substitutions produced complex kinetic changes that together suggested that geometry made a larger contribution than charge. Current decay at positive potentials (O-->I) exponentially approached a constant value for all mutants except R2K channels, which were biphasically dependent on potential. R2K channels also displayed slowed deactivation with reduced voltage dependence despite near control values for conductance. Voltage-dependent accessibility of R to C mutants, evaluated with intracellularly and extracellularly applied methanthiosulfonate (MTS) reagents, showed that both R2 and R3 were exposed only when cells were depolarized, although it was not necessary for channels to open. Together, the data indicate that Domain IV/S4 is an activation domain and is not involved in inactivation from the open state.
Mibefradil is a tetralol derivative once marketed to treat hyper-tension. Its primary target is the T-type Ca(2+) channel (IC(50), approximately 0.1-0.2 microM), but it also blocks Na(+),K(+),Cl(-), ...and other Ca(2+) channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na(+) channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Na(v)1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca(2+) channel-like mibefradil sensitivity to the Na(+) channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.
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