Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules.

In this review we discuss and compare recently introduced molecules that are able to react covalently with an oncogenic mutant of KRas, KRas G12C. Two different classes of compounds in question have been developed, both leading to the mutant being locked in the inactive (guanosine diphosphate [GDP]-bound) state. The first are compounds that interact reversibly with the switch-II pocket (S-IIP) before covalent interaction. The second class interact in a competitive manner with the GDP/guanosine triphosphate (GTP) binding site. The fundamental physico-chemical principles of the two inhibitor classes are evaluated. For GDP/GTP-competing molecules, we show that special attention must be paid to the influence of guanine nucleotide exchange factors (GEFs) and their elevated activity in cells harboring abnormally activated Ras mutants. A new approach is suggested involving compounds that interact with the guanine binding site of the GTPase, but in a manner that is independent of the interaction of the GTPase with its cognate GEF.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

Synthesis and SAR study of novel 3,3-diphenyl-1,3-dihydroindol-2-one derivatives as potent eIF2·GTP·Met-tRNAiMet ternary complex inhibitors.

The growing recognition of inhibition of translation initiation as a new and promising paradigm for mechanism-based anti-cancer therapeutics is driving the development of potent, specific, and druggable inhibitors. The 3,3-diaryloxindoles were recently reported as potential inhibitors of the eIF2·GTP·Met-tRNAi(Met) ternary complex assembly and 3-{5-tert-butyl-2-hydroxyphenyl}-3-phenyl-1,3-dihydro-2H-indol-2-one #1181 was identified as the prototypic agent of this chemotype. Herein, we report our continuous effort to further develop this chemotype by exploring the structural latitude toward different polar and hydrophobic substitutions. Many of the novel compounds are more potent than the parent compound in the dual luciferase ternary complex reporter assay, activate downstream effectors of reduced ternary complex abundance, and inhibit cancer cell proliferation in the low µM range. Moreover, some of these compounds are decorated with substituents that are known to endow favorable physicochemical properties and as such are good candidates for evaluation in animal models of human cancer.

Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

A novel nucleoside analog, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro.

Antiviral therapies are urgently needed to control emerging flaviviruses such as dengue, West Nile, and yellow fever. Ribavirin (RBV) has shown activity against flaviviruses in cultured cells, but efficacy in animal models has generally been poor. In a preliminary screen of novel, synthetic 1-beta-d-ribofuranosyl-azole analogs, two compounds, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR) and 1-beta-d-ribofuranosyl-4-ethynyl-[1,3]imidazole (IM18), significantly reduced the replication of dengue virus serotype 2 (DENV-2) in cultured Vero cells. In the current study we demonstrated that the effective concentration 50 (EC(50)) of ETAR for DENV-2 is substantially lower than both IM18 and RBV. Moreover, ETAR reduced the replication of five additional flaviviruses, including DENV serotypes 1, 3 and 4, Langat virus and Modoc virus, > or =1000-fold relative to untreated controls. Addition of exogenous guanosine to DENV-2 infected cells negated the antiviral effects of both RBV and ETAR, indicating that GTP depletion is a major mechanism of action for both drugs. ETAR represents a promising drug candidate for the treatment of flavivirus infections.

Synthesis of 1-beta-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus.

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

Effect of inhibition of farnesylation and geranylgeranylation on renal fibrogenesis in vitro.

BACKGROUND: The Ras and Rho family of GTPases serve as essential molecular switches in the downstream signalling of many cytokines involved in the regulation of renal fibroblast activity. Prenylation is a post-translational process critical to the membrane localization and function of these GTPases. We studied the effects of a farnesyltransferase inhibitor BMS-191563 and geranylgeranyltransferase inhibitor GGTI-298 on renal fibrogenesis in vitro. METHODS: Functional studies examined the effects of BMS-191563 and GGTI-298 on rat renal fibroblast kinetics, collagen synthesis and collagen gel contraction. Pro-collagen alpha1(I) mRNA expression was measured by Northern analysis and CTGF expression by Western blotting. RESULTS: Fibroblast proliferation was significantly reduced by both agents. Exposure of fibroblasts to BMS-191563 resulted in a significant reduction in total collagen production and pro-collagen alpha1(I) mRNA expression, an effect also observed but to a lesser degree with GGTI-298. Both agents significantly reduced CTGF protein expression. Fibroblast-mediated collagen I lattice contraction was decreased at 48 h by GGTI-298, an effect not observed with BMS-191563. Consistent with this finding, marked actin filament disassembly was evident by phalloidin staining of fibroblasts exposed to GGTI-298. CONCLUSION: BMS-191563 and GGTI-298 exhibit different effects on renal fibroblast function reflecting their predominant roles in inhibiting prenylation of Ras or Rho proteins respectively. Further studies are warranted to establish their potential therapeutic application in the treatment of progressive renal disease.

Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin.

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.

Mechanisms involved in carbachol-induced Ca(2+) sensitization of contractile elements in rat proximal and distal colon.

1. Mechanisms involved in Ca(2+) sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2. In alpha-toxin-permeabilized preparations from the rat proximal and distal colon, Ca(2+) induced a rapid phasic and subsequent tonic component. After Ca(2+)-induced contraction reached a plateau, guanosine 5'-triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca(2+) sensitization). Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization more significantly in the proximal colon than in the distal colon. 3. Y-27632 at 10 microm had no effect on Ca(2+)-induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca(2+) sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization in the distal colon, but not in the proximal colon. The component of Ca(2+) sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4. In beta-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca(2+) sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca(2+) sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5. These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca(2+) sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.

Galectin-1(L11A) predicted from a computed galectin-1 farnesyl-binding pocket selectively inhibits Ras-GTP.

Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.

Phospholipase C, but not InsP3 or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells.

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

Concerning the chemical nature of tubulin subunits that cap and stabilize microtubules.

There is no definitive evidence on the nature of the cap at microtubule ends that is responsible for dynamic instability behavior. It was, therefore, of interest that steady-state microtubules assembled in 20 mM P(i) buffer and pulsed for 15-60 min with [gamma-(32)P]GTP contained approximately 26 [(32)P]P(i)/microtubule [Panda et al. (2002) Biochemistry 41, 1609-1617]. It was concluded that microtubules are capped with a tubulin-GDP-P(i) subunit at the end of each its 13 protofilaments and that this is responsible for stabilizing microtubules in the growth phase. Also, because microtubules with [(32)P]P(i) were isolated despite the presence of 20 mM P(i), it was concluded that P(i) in terminal tubulin-GDP-P(i) subunits does not exchange with solvent. These observations are inconsistent with our finding that tubulin-GDP-P(i) subunits do not stabilize microtubules and with evidence that the nucleotide, and presumably also P(i), in subunits at microtubule ends exchanges with solvent. We have resolved this discrepancy by finding that during the pulse period the added [(32)P]GTP was almost quantitatively hydrolyzed. The so-formed [(32)P]P(i) labeled the 20 mM P(i) buffer, and this exchanged into tubulin-GDP subunits in the core of the microtubule. Evidence for this was our finding of virtually identical [(32)P]P(i) in microtubules pulsed with [(32)P]GTP with a specific activity that varied 11-fold by using either 100 or 1,100 microM GTP in the reaction. Label uptake was insensitive to the [(32)P]GTP specific activity because in both cases hydrolysis generated 20 mM [(32)P]P(i) with a virtually identical specific activity. Also, approximately 0.4 mol of [(32)P]P(i) /tubulin dimer was found in microtubules when steady-state microtubules in 20 mM P(i) were pulsed with a trace amount of [(32)P]P(i). This stoichiometry is consistent with a 25 mM K(d) previously reported for P(i) binding to tubulin-GDP subunits in microtubules. It is concluded that, under the conditions used for the [(32)P]GTP pulse labeling, (32)P was incorporated into the entire microtubule from [(32)P]P(i) released into the solution, rather than into a tubulin-GDP-P(i) cap, from [(32)P]GTP. Thus, there is no evidence that tubulin-GDP-P(i) subunits accumulate in and stabilize microtubule ends.

Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs.

A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP >> Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.

Characterization of the interaction of TZT-1027, a potent antitumor agent, with tubulin.

TZT-1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT-1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT-1027 and dolastatin 10 inhibited microtubule polymerization concentration-dependently at 1 - 100 microM with IC50 values of 2.2 +/- 0.6 and 2.3 +/- 0.7 microM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1 - 3 microM with IC50 values of 2.7 +/- 0.6, 1.6 +/- 0.4 and 1.6 +/- 0.2 microM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 microM or more. TZT-1027 also inhibited monosodium glutamate-induced tubulin polymerization concentration-dependently at 0.3 - 10 microM, with an IC50 of 1.2 microM, whereas VLB was only effective at 0.3 - 3 microM, with an IC50 of 0.6 microM, and caused so-called "aggregation" of tubulin at 10 microM. Scatchard analysis of the binding data for [(3)H]VLB suggested one binding site (Kd 0.2 +/- 0.04 microM and Bmax 6.0 +/- 0.26 nM / mg protein), while that for [(3)H]TZT-1027 suggested two binding sites, one of high affinity (Kd 0.2 +/- 0.01 microM and Bmax 1.7 +/- 0.012 nM / mg protein) and the other of low affinity (Kd 10. 3 +/- 1.46 microM and Bmax 11.6 +/- 0.83 nM / mg protein). [(3)H]TZT-1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [(3)H]VLB was completely displaced by dolastatin 10 and TZT-1027. Furthermore, TZT-1027 prevented [(3)H]VLB from binding to tubulin in a non-competitive manner according to Lineweaver-Burk analysis. TZT-1027 concentration-dependently inhibited both [(3)H]guanosine 5'-triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration-dependently to a lesser extent than TZT-1027, but no inhibitory effect of VLB on [(3)H]GTP binding to tubulin was evident even at 100 microM. Thus, TZT-1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT-1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.

Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes.

BACKGROUND: Mycophenolate mofetil (MMF) is an effective immunosuppressant developed for use in organ transplantation. It specifically targets lymphocyte purine biosynthesis. However, side effects do occur. Understanding how the active metabolite of MMF, mycophenolic acid (MPA) affects the normally integrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes. METHODS: We used a primary human T-lymphocyte model to study how preincubation with MPA (0.1-50 microM) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors. RESULTS: MPA not only restricted the mitogen-induced expansion of GTP pools, but actually induced a severe drop in both GTP (10% of unstimulated cells) and GDP-sugar pools, with a concomitant fall in ATP (up to 50%). These effects were concentration dependent. By contrast, uridine pools expanded whereas CTP pools remained at resting levels. These changes were confirmed by the altered incorporation of [14C]-bicarbonate and [14C]-glycine into nucleotides. Restriction of [14C]-hypoxanthine incorporation and reduction of [14C]-uridine uptake comparable to that of unstimulated cells indicated that MPA also inhibited both salvage routes of nucleotide synthesis. CONCLUSION: MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies.

Inhibition of Ras and related guanosine triphosphate-dependent proteins as a therapeutic strategy for blocking malignant glioma growth: II--preclinical studies in a nude mouse model.

OBJECTIVE: Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems. METHODS: We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test. RESULTS: Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001). CONCLUSION: These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Fatty acid-mediated calcium sequestration within intracellular calcium pools.

Intracellular Ca2+ pools play an essential role in generating Ca2+ signals. The heterogeneity of intracellular Ca2+ pools reflects the complex and dynamic character of the endoplasmic reticulum within which they reside. Translocation of Ca2+ between distinct subcompartments of the endoplasmic reticulum is mediated by a sensitive and specific GTP-activated process involving formation of reversible communicating junctions (Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613). In the presence of palmitate at 10 microM or above, this GTP-activated mechanism mediates substantial Ca2+ accumulation within a specific Ca2+-pumping pool. The fatty acid- and GTP-dependent accumulation of Ca2+ was highly chain length-specific; pentadecanoate (C15) and palmitate (C16) were equally effective, whereas fatty acids of shorter or longer chain length were either marginally effective or devoid of effect. Fatty acids with one or more unsaturated carbons were without effect, regardless of chain length. Palmitate-induced Ca2+ accumulation was immediately terminated with 2 microM palmitoyl-CoA, a blocker of the GTP-activated Ca2+-translocating mechanism. The anion transport inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid completely prevented both palmitate- and oxalate-mediated GTP-dependent Ca2+ accumulation, with EC50 approximately 30 microM. Ca2+ sequestered in the presence of palmitate and GTP could be immediately and completely released by A23187, whereas the sequestered Ca2+ was remarkably resistant to release induced by inositol 1,4,5-trisphosphate (InsP3). In contrast, oxalate-sequestered Ca2+ within the same pool could be effectively released by either ionophore or InsP3. The results indicate that fatty acids are specifically transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate that is not readily releasable by opened InsP3-sensitive Ca2+ channels.

Purine metabolism and immunosuppressive effects of mycophenolate mofetil (MMF).

Mycophenolate mofetil (MMF) is a novel immunosuppressive drug that shows promise in preventing the rejection of organ allografts and in the treatment of ongoing rejection. Orally administered MMF is hydrolyzed by esterases in the intestine and blood to release mycophenolic acid (MPA), a potent, selective, noncompetitive inhibitor of the type 2 isoform of inosine monophosphate dehydroxygenase (IMPDH) expressed in activated human T and B lymphocytes. By inhibiting IMPDH, MPA depletes the pool of dGTP required for DNA synthesis. MPA has a more potent cytostatic effect on lymphocytes than on other cell types, and this is the principal mechanism by which immunosuppressive activity is exerted. MPA also depletes pools of GTP in human lymphocytes and monocytes, thereby inhibiting the synthesis of fucose- and mannose-containing saccharide components of membrane glycoproteins. These are recognized by the family of adhesion molecules termed selectins. By this mechanism, MPA could decrease the recruitment of lymphocytes and monocytes into sites of graft rejection. In addition to preventing allograft rejection, MMF suppresses graft-versus-host reactions in lethal and nonlethal murine models. MMF inhibits primary antibody responses more efficiently than secondary responses. MPA inhibits the proliferation of human B lymphocytes transformed by Epstein-Barr virus and is not mutagenic. Clinically attainable concentrations of MPA suppress the proliferation of human arterial smooth muscle cells. These two properties of MPA may decrease the risk of lymphoma development and proliferative arteriopathy in long-term recipients of MMF.