Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.
Estrogens can potentially be classified into planar (Class I) or non planar (Class II) categories, which might have biological consequences. 1,1,2-Triphenoplsylethylene (TPE) derivatives were synthesized and evaluated against 17β-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer cells. All TPEs were estrogenic and unlike 4-hydroxytamoxifen (4OHTAM) and endoxifen, induced cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells with the order of potency as followed: E2 > 1,1-Bis(4,4′-hydroxyphenyl)-2-phenylbut-1-ene (15) > 1,1,2-Tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenol (12) > 4-OHTAM. Transient transfection of the ER-negative breast cancer cell line T47D:C4:2 with wildtype ER or D351G ER mutant revealed that all of the TPEs increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a Class I estrogen and the TPEs as Class II estrogens. Using available conformations of the ER liganded with 4OHTAM or diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.
5-Chloro[1,4-13C]levulinic acid ([1,4-13C]CLA) is an active site-directed inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrically condenses two molecules of 5-aminolevulinic acid (ALA) which are called A-side ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. [1,4-13C]CLA modifies bovine PBGS at the A-side ALA binding site. The C4 chemical shift indicates an intact keto moiety; the C1 chemical shift indicates a deprotonated carboxyl group. In contrast, [1,4-13C]CLA modification of Escherichia coli PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C1 chemical shifts indicate substantially deprotonated carboxylic acid groups. For one of four observed forms of [1,4-13C]CLA-modified E. coli PBGS, an analog of the P-site Schiff base is found. Bovine and E. coli PBGS contain two different zincs, ZnA and ZnB. Past results placed ZnA near A-side ALA. [1,4-13C]CLA modifies E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine cluster implicated in binding ZnB. This result places ZnB near P-side ALA. E. coli PBGS binds a third type of divalent metal, MgC or MnC, which is found to have no significant effect on the 13C NMR spectrum of the [1,4-13C]CLA-modified protein.
Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.
The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), ( 1-oxyl-2,2,5,5-tetramethyl-A3 -pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L.J., Grunwald, J., Hankovszky, H.O., & Hideg, K., 1982, Anal. Biochem. 119,450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (Am = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions shows that at least two of the unpaired electrons are within at most 7.6 A of each other.SL-MMTS-modified PBGS loses all Zn(1I) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added. The results indicate that binding of this substrate does not affect the environment of the majority of the nitroxides. This is consistent with our previous proposal that it is the substrate that binds only to the Zn(I1) enzyme that provides ligands to the catalytic Zn(I1) (Jaffe, E.K., Abrams, W.R.,, Kaempfen, K.X., & Harris, K.A., 1992, Biochemistry 31, 2113-2123).SL-MMTS readily forms disulfide-linked dimers. The rate of dimer formation is proportional to the hydroxide ion concentration and is independent of both buffer and SL-MMTS concentrations. This suggests a ratelimiting hydrolysis of the s-S bond of SL-MMTS followed by rapid reaction of the liberated thiol with a second molecule of SL-MMTS. Therefore, substantial care is required in the use of SL-MMTS as a protein modification reagent at even slightly alkaline pH values.Keywords: porphobilinogen synthase; spin-labeled cysteine; spin label MMTS dimerization Porphobilinogen synthase catalyzes the condensation of two molecules of ALA t o form PBG, the monopyrrole precursor to all naturally occurring tetrapyrroles (e.g., Abbreviations: ALA, 5-aminolevulinate; EXAFS, extended X-ray absorption fine structure; HPLC, high-performance liquid chromatography; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid; BME, 2-mercaptoethanol; MMTS, methyl methanethiosulfonate; KPi, potassium phosphate; SL-MMTS, (I-oxyl-2,2,5,5-tetramethyl-A3-pyrroline-3-m...
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