Current Role at Stanford
Responsible for the scientific service centers in the School of Medicine.
Adviser to the SPARK Program
Responsible for the scientific service centers in the School of Medicine.
Adviser to the SPARK Program
Promiscuous binders achieve enzyme inhibition using a nonspecific aggregation-type binding mechanism to proteins. These compounds are a source of false-positive hits in biochemical inhibition assays and should be removed from screening hit lists because they are not good candidates to initiate medicinal chemistry programs. We introduce a robust approach to identify these molecules early in the lead generation process using real time surface plasmon resonance based biosensors to observe the behavior of the binding interactions between promiscuous compounds and proteins. Furthermore, the time resolution of the assay reveals a number of distinct mechanisms that promiscuous compounds employ to inhibit enzyme function and indicate that the type of mechanism can vary depending on the protein target. A classification scheme for these compounds is presented that can be used to rapidly characterize the hits from high-throughput screens and eliminate compounds with a nonspecific mechanism of inhibition.
View details for DOI 10.1021/jm700952v
View details for Web of Science ID 000252968700026
View details for PubMedID 18181566
Neuropathic pain may be produced, at least in part, by the increased activity of primary afferent neurons. Studies have suggested that an accumulation of voltage-gated sodium channels at the site of peripheral nerve injury is a primary precursory event for subsequent afferent hyperexcitability. In this study, a human sodium channel (hPN3, SCN10A) has been cloned from the lumbar 4/5 dorsal root ganglia (DRG). Expression of hPN3 in Xenopus oocytes showed that this clone is a functional voltage-gated sodium channel. The amino acid sequence of hPN3 is most closely related to the rat PN3/SNS sodium channels which are expressed primarily in the small neurons of rat DRGs. The homologous relationship between rPN3 and hPN3 is defined by (i) a high level of sequence identity (ii) sodium currents that are highly resistant to tetrodotoxin (TTX) (iii) similar tissue distribution profiles and (iv) orthologous chromosomal map positions. Since rPN3/SNS has been implicated in nociceptive transmission, hPN3 may prove to be a valuable target for therapeutic agents against neuropathic pain.
View details for Web of Science ID 000077042900003
View details for PubMedID 9839820
Neurons of the dorsal root ganglia (DRG) express a diversity of voltage-gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin-sensitive sodium channel alpha subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT-PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low-level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V(1/2) approximately -100 mV for steady-state inactivation and V(1/2) approximately -40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50 approximately 1 nM. Coexpression of the beta1 subunit accelerated inactivation kinetics, but the beta2 subunit was without effect.
View details for Web of Science ID 000073647800004
View details for PubMedID 9603190
Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 >/= PN3 > rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 +/- 0.92 nM. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the beta-subunits.
View details for Web of Science ID A1997XC32700045
View details for PubMedID 9169448
Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.
View details for Web of Science ID A1996UA36300001
View details for PubMedID 8626372
In HL-60 cells, inhibition of the endoplasmic-reticular Ca2+ pump by thapsigargin leads to the emptying of this intracellular Ca2+ store and a subsequent activation of plasma-membrane Ca2+ influx through a non-voltage-dependent pathway. The elevated intracellular free Ca2+ concentration ([Ca2+]i) produced and maintained by this Ca2+ inflow was used to examine the potency of various compounds to inhibit this influx mechanism. As expected, specific blockers of known Ca2+ channels, such as nifedipine, omega-conotoxin GVIA and ryanodine were without effect. The less selective inhibitors La3+, SKF-96365 and L-651,582, which are thought to inhibit both voltage-dependent and voltage-independent Ca2+ channels, decreased [Ca2+]i back to resting levels, with pIC50 values of 5.2, 5.9 and 6.2 respectively. It has been proposed that a cytochrome P-450 is involved in activating Ca(2+)-influx pathways in thymocytes, neutrophils and platelets. Consistent with this idea, the imidazole cytochrome P-450 inhibitors miconazole, econazole, clotrimazole and ketoconazole inhibited the thapsigargin-elevated [Ca2+]i with pIC50 values of 7.1, 7.1, 7.1 and 5.8 respectively. The high affinity of imidazoles for cytochromes P-450 is due to co-ordinate binding to the haem. This interaction is greatly decreased in 2-substituted imidazoles. We examined whether the inhibition of Ca2+ influx was due to an interaction of the inhibitor imidazole nitrogen with the haem iron of the putative cytochrome P-450 by comparing the activity of two compounds, identical except that one was methylated at the imidazole 2-position. They were found to block thapsigargin-activated Ca2+ influx with equal potency. These results strongly suggest that a cytochrome P-450 is not involved in the activation of the Ca2+ influx produced by emptying the intracellular Ca2+ stores.
View details for Web of Science ID A1994PC82800027
View details for PubMedID 8068004
Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.
View details for Web of Science ID A1988N136900052
View details for PubMedID 3282178
The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.
View details for Web of Science ID A1988L504700037
View details for PubMedID 2891695
The neuropeptide somatostatin inhibits prolactin release from GH4C1 pituitary cells via two mechanisms, inhibition of stimulated adenylate cyclase activity and an undefined cAMP-independent process. Somatostatin also hyperpolarizes GH4C1 cells and reduces their intracellular free Ca2+ concentration ([Ca2+]i) in a cAMP-independent manner. To determine whether these ionic changes were involved in the cAMP-independent mechanism by which somatostatin inhibited secretion, changes in cAMP levels were prevented from having any biological consequences by performing experiments in the presence of a maximal concentration of a cAMP analog. Under these conditions, inhibition of prolactin release by somatostatin required a transmembrane concentration gradient for K+ but not one for either Na+ or Cl-. However, elimination of the outward K+ gradient did not prevent somatostatin inhibition of vasoactive intestinal peptide-stimulated hormone release. Therefore, somatostatin's cAMP-mediated mechanism does not require a K+ gradient, whereas its cAMP-independent inhibition of secretion appears to result from a change in K+ conductance. Consistent with this conclusion, membrane hyperpolarization with gramicidin (1 microgram/ml) mimicked somatostatin inhibition of prolactin release. In addition, the K+ channel blocker tetrabutylammonium prevented the effects of somatostatin on the membrane potential, the [Ca2+]i and hormone secretion. Nonetheless, a K+ gradient was not sufficient for somatostatin action. Even in the presence of a normal K+ gradient, somatostatin was only able to inhibit prolactin release when the extracellular Ca2+ concentration was at least twice the [Ca2+]i. Furthermore, the calcium channel blocker, nifedipine (10 microM), which prevents the action of somatostatin to reduce the [Ca2+]i, specifically blocked inhibition of prolactin release via somatostatin's cAMP-independent mechanisms. Therefore, a decrease in Ca2+ influx through voltage-dependent Ca2+ channels produces both the fall in [Ca2+]i and inhibition of hormone secretion in response to somatostatin.
View details for Web of Science ID A1988L504700038
View details for PubMedID 2891696
The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.
View details for PubMedID 2865257
We have examined the mechanisms by which S-14 inhibits pituitary hormone secretion in a homogeneous cell population: the clonal GH4C1 cell line. The S-14 receptor in GH4C1 cells is coupled to Ni, a guanine nucleotide binding protein which mediates S-14-induced inhibition of VIP-stimulated adenylate cyclase activity, cyclic AMP production and hormone secretion. In addition, a functional Ni is required for S-14 to inhibit basal hormone secretion, an action which appears to be independent of cyclic AMP concentrations. Accumulating evidence indicates that the mechanism of S-14 action in somatotrophs is similar to that in GH4C1 cells. Although S-14 consistently inhibits basal GH secretion, its effects on basal cyclic AMP levels in normal pituitary cells are variable and often not significant (10-14). In contrast, S-14 inhibits prostaglandin and growth hormone releasing factor (GRF) stimulated cyclic AMP accumulation and GH release in parallel. Furthermore, S-14 partially blocks prostaglandin and GRF stimulation of adenylate cyclase activity in rat anterior pituitary membranes. Finally, pretreatment of primary cultures of rat pituitary cells with IAP antagonizes S-14 inhibition of both basal and GRF-stimulated GH release.
View details for Web of Science ID A1985ANF1500017
View details for PubMedID 2863938
Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. In this study we examined the interaction between the SRIF receptor and adenylate cyclase in GH4C1 cell membranes. Maximal concentrations of VIP (50 nM) increased membrane adenylate cyclase activity 4.2-fold; half-maximal stimulation was observed with 0.75 nM VIP. SRIF noncompetitively inhibited the stimulatory effect of VIP, but it did not alter basal adenylate cyclase activity. The relative potencies of SRIF and two SRIF analogs as inhibitors of VIP-stimulated adenylate cyclase activity in membranes and of VIP-stimulated cAMP accumulation in intact cells were similar. Furthermore, the concentration of SRIF that caused half-maximal inhibition of adenylate cyclase activity (ED50 = 2.3 nM) was close to the equilibrium dissociation constant for SRIF (Kd = 0.40 nM) measured in membrane preparations in the presence of GTP. Therefore, SRIF inhibition of adenylate cyclase appears to be receptor mediated. As with receptors known to regulate adenylate cyclase by interaction with a guanine nucleotide regulatory subunit, SRIF receptor binding was decreased in the presence of guanine nucleotides. Addition of GTP (150 microM) or the nonhydrolyzable GTP analog guanyl-5'-yl-imidodiphosphate (100 microM) decreased the specific binding of [125I-Tyr1]SRIF to 31% and 13% of the control value, respectively. This decrease in specific binding was due entirely to decreased receptor affinity for SRIF. GTP (150 microM) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM, whereas the number of binding sites was unaffected by the nucleotide (Bmax = 0.2 pmol/mg protein). Analysis of dissociation kinetics demonstrated that in the absence of guanyl nucleotides, the rate of [125I-Tyr1]SRIF dissociation was first order (t 1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5'-yl-imidodiphosphate (0.3 microM), [125I-Tyr1]SRIF dissociation occurred with biphasic kinetics. Fifty percent of the specifically bound peptide dissociated at the same rate as that observed in the absence of nucleotide, whereas the remainder dissociated 15 times more rapidly (t 1/2 = 9.6 min).(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1984SP24200043
View details for PubMedID 6143660
The effects of temperature in the fever range (37-42 degrees) on the membrane potentials of normal and simian virus 40-transformed hamster lymphocytes were analyzed. The transmembrane distributions of radiolabeled triphenylmethylphosphonium and thiocyanate were measured, and they provide upper and lower limits for the normal cell membrane potential at 37 degrees of -48 +/- 6 (S.D.) and -31 +/- 5 mV and for the tumor cells, -36 +/- 4 and -19 +/- 2 mV. The mitochondrial contribution to the triphenylmethylphosphonium-measured membrane potential, 5 to 10 mV for both splenocytes and simian virus 40-transformed lymphocytes, was estimated by utilizing antimycin A and carbonylcyanide-m-chlorophenylhydrazone to inhibit generation of a mitochondrial membrane potential. Incubation for 1 to 2 hr at 38-42 degrees resulted in a 6- to 15-mV depolarization of normal cells and a 2- to 6-mV hyperpolarization of tumor cells. Both depolarization and hyperpolarization were fully reversible by subsequent incubation at 37 degrees and insensitive to antimycin A and carbonyl-cyanide-m-chlorophenylhydrazone. The membrane potential of normal splenocytes when measured with triphenylmethylphosphonium at 37 degrees was depolarized by 35% with 1 mM ouabain and thermally induced depolarization was blocked. The membrane potential of tumor cells at 37 degrees was insensitive to ouabain; however, the hyperpolarization at 40 degrees was inhibited. The membrane potential of normal lymphocytes stimulated with phytohemagglutinin was depolarized relative to that of nonstimulated control cells and assumed the thermal response characteristics of tumor cells.
View details for Web of Science ID A1981KX18600035
View details for PubMedID 6256060