Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Design, synthesis and biological evaluation of 5$\sp\prime$-mononucleotide prodrugs: Strategies to introduce nucleotides into cells

The neutral bis ((pivaloyloxy)methyl) (PIV$\sb2\rbrack$ derivatives of FdUMP, ddUMP, and AZTMP were synthesized as potential membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. These compounds were designed to enter cells by passive diffusion and revert to the parent nucleotides after removal of the PIV groups by hydrolytic enzymes. These prodrugs were prepared by condensation of FUdR, ddU, and AZT with PIV$\sb2$ phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP were stable in the pH range 1.0-4.0 (t$\sb{1/2} = {>}$100 h). They were also fairly stable at pH 7.4 (t$\sb{1/2} = {>}$40 h). In 0.05 M NaOH solution, however, they were rapidly degraded (t$\sb{1/2} < 2$ min). In the presence hog liver carboxylate esterase, they were converted quantitatively to the corresponding phosphodiesters, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP; after 24 h incubation, only trace amounts of FdUMP, ddUMP, and AZTMP (1-5%) were observed indicating that the PIV$\sb1$ compounds were poor substrates for the enzyme. In human plasma, the PIV$\sb2$ compounds were rapidly degraded with half-lives of less than 5 min. The rate of degradation of the PIV$\sb2$ compounds in the presence of phosphodiesterase I was the same as that in buffer controls, indicating that they were not substrates for this enzyme. In the presence of phosphodiesterase I, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP were converted quantitatively to FdUMP, ddUMP, and AZTMP.^ PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were effective at controlling HIV type 1 infection in MT-4 and CEM tk$\sp-$ cells in culture. Mechanistic studies demonstrated that PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were taken up by the cells and converted to ddUTP and AZTTP, both potent inhibitors of HIV reverse transcriptase. However, a potential shortcoming of PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP as clinical therapeutic agents is that they are rapidly degraded (t$\sb{1/2}$ = approx. 4 minutes) in human plasma by carboxylate esterases. To circumvent this limitation, chemically-labile nucleotide prodrugs and liposome-encapsulated nucleotide prodrugs were investigated. In the former approach, the protective groups bis(N, N-(dimethyl)carbamoyloxymethyl) (DM$\sb2$) and bis (N-(piperidino)carbamoyloxymethyl) (DP$\sb2$) were used to synthesize DM$\sb2$-ddUMP and DP$\sb2$-ddUMP, respectively. In aqueous buffers (pH range 1.0-9.0) these compounds were degraded with half-lives of 3 to 4 h. They had similar half-lives in human plasma demonstrating that they were resistant to esterase-mediated cleavage. However, neither compound gave rise to significant concentrations of ddUMP in CEM or CEM tk$\sp-$ cells. In the liposome-encapsulated nucleotide prodrug approach, three different liposomal formulations of PIV$\sb2$-ddUMP (L-PIV$\sb2$-ddUMP) were investigated. The half-lifes of these L-PIV$\sb2$-ddUMP preparations in human plasma were 2 h compared with 4 min for the free drug. The preparations were more effective at controlling HIV-1 infection than free PIV$\sb2$-ddUMP in human T cells in culture. Collectively, these data indicate that PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP are effective membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. ^

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^