Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.

Photochemical and Pharmacokinetic Characterization of Orally Administered Chemicals to Evaluate Phototoxic Risk.

This study aimed to verify the applicability of a proposed photosafety screening system based on a reactive oxygen species (ROS) assay and a cassette-dosing pharmacokinetic (PK) study to chemicals with wide structural diversity. The orally taken chemicals, erythromycin, gatifloxacin, 8-methoxypsoralen (MOP), pirfenidone (PFD), trifluoperazine (TFP), and voriconazole (VRZ), were selected as test compounds. The ROS assay was conducted to evaluate their photoreactivity, and all test compounds excluding erythromycin generated significant ROS under simulated sunlight exposure. According to the ROS data, TFP had potent photoreactivity, and the photoreactivity of 4 other compounds was judged to be moderate. Regarding the oral cassette-dosing PK test in rats, the skin deposition of MOP, PFD, and VRZ was relatively high, and gatifloxacin and TFP exhibited moderate skin deposition properties. Based on the ROS and PK data of test compounds, PFD and TFP were judged to be potent phototoxic compounds, and MOP and VRZ were deduced to have phototoxic risk. The predicted phototoxic risk of test compounds by proposed screening was mostly in agreement with observed in vivo phototoxicity in the rat skin. The proposed screening system could provide reliable photosafety information on orally administered compounds with wide structural diversity.

Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies.

Patau Syndrome (PS), characterized as a lethal disease, allows less than 15% survival over the first year of life. Most deaths owe to brain and heart disorders, more so due to septal defects because of altered gene regulations. We ascertained the cytogenetic basis of PS first, followed by molecular analysis and docking studies. Thirty-seven PS cases were referred from the Department of Pediatrics, King Abdulaziz University Hospital to the Center of Excellence in Genomic Medicine Research, Jeddah during 2008 to 2018. Cytogenetic analyses were performed by standard G-band method and trisomy13 were found in all the PS cases. Studies have suggested that genes of chromosome 13 and other chromosomes are associated with PS. We, therefore, did molecular pathway analysis, gene interaction, and ontology studies to identify their associations. Genomic analysis revealed important chr13 genes such as FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, and TUBA3D, and their regulatory partners on other chromosomes associated with cardiovascular disorders, atrial and ventricular septal defects. There is strong indication of involving FOXO1 (Forkhead Box O1) gene-a strong transcription factor present on chr13, interacting with many septal defects link genes. The study was extended using molecular docking to find a potential drug lead for overexpressed FOXO1 inhibition. The phenothiazine and trifluoperazine showed efficiency to inhibit overexpressed FOXO1 protein, and could be potential drugs for PS/trisomy13 after validation.

Reprint of: A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the 'fuzzy' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

Trifluoperazine blocks the human cardiac sodium channel, Nav1.5, independent of calmodulin.

Trifluoperazine is a phenothiazine derivative which is mainly used in the management of schizophrenia and also acts as a calmodulin inhibitor. We used the whole-cell patch-clamp technique to study the effects of trifluoperazine on human Nav1.5 (hNav1.5) currents expressed in HEK293 cells. The 50% inhibitory concentration of trifluoperazine was 15.5 ± 0.3 µM and the Hill coefficient was 2.7 ± 0.1. The effects of trifluoperazine on hNav1.5 were completely and repeatedly reversible after washout. Trifluoperazine caused depolarizing shifts in the activation and hyperpolarizing shifts in the steady-state inactivation of hNav1.5. Trifluoperazine also showed strong use-dependent inhibition of hNav1.5. The blockade of hNav1.5 currents by trifluoperazine was not affected by the whole cell dialysis of the calmodulin inhibitory peptide. Our results indicated that trifluoperazine blocks hNav1.5 current in concentration-, state- and use-dependent manners rather than via calmodulin inhibition.

Ganoderic acid B's influence towards the therapeutic window of trifluoperazine (TFP).

BACKGROUND: Ganoderic acid B is an important bioactive ingredient isolated from Ganoderma lucidum, and exhibits various pharmacological activities. AIMS: To investigate the influence of Ganoderic acid B towards the therapeutic window of trifluoperazine (TFP). METHODS: In vitro human liver microsomes (HLMs) incubation system was used to determine the inhibition of Ganoderic acid B towards the glucuronidation of trifluoperazine (TFP). RESULTS: Ganoderic acid B exerted concentration-dependent inhibition towards the glucuronidation of TFP. Furthermore, Dixon plot was used to determine the inhibition type. The intersection point was located in the second quadrant in Dixon plot, indicating the competitive inhibition of Ganoderic acid B towards TFP glucuronidation. Through fitting the data using competitive nonlinear fitting equation, the inhibition kinetic parameter was calculated to be 56.7 uM. CONCLUSION: All this data indicated the potential influence of Ganoderic acid B-containing herbs towards therapeutic window of TFP. Given that the glucuronidation reaction of TFP is the probe reaction of UGT1A4, the data obtained from the present study also indicated the potential influence of Ganoderic acid-containing herbs towards the therapeutic window of drugs mainly undergoing UGT1A4-mediated metabolism.

Effect of efavirenz on UDP-glucuronosyltransferase 1A1, 1A4, 1A6, and 1A9 activities in human liver microsomes.

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for the treatment of human immunodeficiency virus type 1 infections. Drug interactions of efavirenz have been reported due to in vitro inhibition of CYP2C9, CYP2C19, CYP3A4, and UDP-glucuronosyltransferase 2B7 (UGT2B7) and in vivo CYP3A4 induction. The inhibitory potentials of efavirenz on the enzyme activities of four major UDP-glucuronosyltransferases (UGTs), 1A1, 1A4, 1A6, and 1A9, in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Efavirenz potently inhibited UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation, with K(i) values of 2.0 and 9.4 µM, respectively. [I]/K(i) ratios of efavirenz for trifluoperazine N-glucuronidation and propofol glucuronidation were 6.5 and 1.37, respectively. Efavirenz also moderately inhibited UGT1A1-mediated 17ß-estradiol 3-glucuronidation, with a K(i) value of 40.3 µM, but did not inhibit UGT1A6-mediated 1-naphthol glucuronidation. Those in vitro results suggest that efavirenz should be examined for potential pharmacokinetic drug interactions in vivo due to strong inhibition of UGT1A4 and UGT1A9.

Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I.

The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.

Synthesis, biological evaluation, and docking studies of gigantol analogs as calmodulin inhibitors.

Several analogs of gigantol (1) were synthesized to evaluate their effect on the complexes Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-CaM sensitive phosphodiesterase 1 (PDE1). The compounds belong to four structural groups including, 1,2-diphenylethanes (2-11), diphenylmethanes (13-15), 1,3-diphenylpropenones (16-18), and 1,3-diphenylpropanes (20-22). In vitro enzymatic studies showed that all compounds except 11 inhibited the complex Ca(2+)-CaM-PDE1 with IC(50) values ranging from 9 to 146 µM. On the other hand, all analogs but 11, 12 and 15 quenched the extrinsic fluorescence of the CaM biosensor hCaM-M124C-mBBr to different extent, then revealing different affinities to CaM; their affinity constants (K(m)) values were in the range of 3-80 µM. Molecular modeling studies indicated that all these compounds bound to CaM at the same site that the classical inhibitors trifluoperazine (TFP) and chlorpromazine (CPZ). Some of these analogs could be worthy candidates for developing new anti-tumor, local anesthetics, antidepressants, antipsychotic, or smooth muscle relaxant drugs, with anti-CaM properties due to their good affinity to CaM and the straightforwardness of their synthesis. In addition they could be valuable tools for the study of Ca(2+)-CaM functions.

Phenothiazines inhibit S100A4 function by inducing protein oligomerization.

S100A4, a member of the S100 family of Ca(2+)-binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensor-based assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 A crystal structure of human Ca(2+)-S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca(2+)-S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca(2+)-S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca(2+)-S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.

Measurement of antioxidant activity with trifluoperazine dihydrochloride radical cation

A novel, rapid and cost-effective trifluoperazine dihydrochloride (TFPH) decolorization assay is described for the screening of antioxidant activity. A chromogenic reaction between TFPH and potassium persulfate at low pH produces an orange-red radical cation with maximum absorption at 502 nm in its first-order derivative spectrum. TFPH was dissolved in distilled water to give a 100 mM solution. The TFPH radical cation solution was made by reacting 0.5 mL of the solution with K2S2O8 (final concentration: 0.1 mM) and diluting to 100 mL with 4 M H2SO4 solution. A linear inhibition of color production was observed with linearly increasing amounts of antioxidants, with correlation coefficients (R²) ranging from 0.999 to 0.983. The antioxidant capacity of standard solutions of an antioxidant was evaluated by comparing with the inhibition curve using Trolox as the standard. Comparison of antioxidant capacity determined with this newly developed TFPH assay and with the well-known 2,2'-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS)-persulfate decolorization assay indicated the efficacy and sensitivity of the procedure. The proposed assay is less expensive (costs about US$4 per 100 assays) and requires only 20 min for preparation of radical cation solution in comparison with ABTS assay, in which almost 12-16 h are required for preparation of a stable ABTS radical cation solution. The present assay has the advantage over ABTS assay that it can be used to measure the antioxidant activity of the samples, which are naturally found at a pH as low as 1, because the radical cation itself has been stabilized at low pH.

A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics.

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.

Calmodulin is required for vasopressin-stimulated increase in cyclic AMP production in inner medullary collecting duct.

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug.

3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.

Ca2+ dependence and inhibitory effects of trifluoperazine on plasma membrane ATPase of Thermoactinomyces vulgaris.

Ca2+ enhanced the plasma membrane Ca(2+)-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris. The Ca(2+)-ATPase specific activities showed marked increase with increasing concentrations of Ca2+ added in the form of CaCl2 in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T. vulgaris. Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 microM and 8 microM along with the respective optimal concentrations of Ca2+, decreased the PMCA-specific activities to a low level in a dose-dependent manner. The results of the present investigation suggest the presence of a Ca(2+)-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca(2+)-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete.

Insights into the interactions between a drug and a membrane protein target by fluorine cross-polarization magic angle spinning NMR.

The fluorinated anti-psychotic drug trifluoperazine (TFP) has been shown to be a K(+)-competitive inhibitor of gastric H(+)/K(+)-ATPase, a membrane-embedded therapeutic target for peptic ulcer disease. This paper describes how variable contact time (19)F cross-polarization magic angle spinning (VCT-CP/MAS) NMR has been used to probe the inhibitory interactions between TFP and H(+)/K(+)-ATPase in native gastric membranes. The (19)F CP/MAS spectra for TFP in H(+)/K(+)-ATPase enriched (GI) gastric membranes and in control membranes containing less than 5 nmol of the protein indicated that the drug associates with the membranes independently of the presence of H(+)/K(+)-ATPase. The (19)F peak intensities in the VCT-CP/MAS experiment confirmed that TFP undergoes slow dissociation (k(off) < 100 s(-1)) from binding sites in GI membranes, and more rapid dissociation (k(off) < 100 s(-1)) from control membranes. The spectra showed that up to 40% of bound TFP was displaced from GI membranes by 100 mM K(+) and by the K(+)-competitive inhibitor TMPIP, but TFP was not displaced from the control membranes. Hence the spectra of TFP in GI membranes represent the drug bound to the K(+)-competitive inhibitory site of H(+)/K(+)-ATPase and to other non-specific sites. The affinity of TFP for the K(+)-competitive site (K(D) = 4 mM) was determined from a binding curve of (19)F peak intensity versus TFP concentration after correction for non-specific binding. The K(D) was much higher than the IC(50) for ATPase inhibition (8 microM), which suggests that the substantial non-specific binding of TFP to the membranes contributes to ATPase inhibition. This novel approach to probing ligand binding can be applied to a wide range of membrane-embedded pharmaceutical targets, such as G-protein coupled receptors and ion channels, regardless of the size of the protein or strength of binding.

Pathways involved in trifluoperazine-, dibucaine- and praziquantel-induced hemolysis.

This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.

Molecular modeling of G-protein coupled receptor kinase 2: docking and biochemical evaluation of inhibitors.

G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5'-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 microM (pA26.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 microM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.