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Biomedical subjects

A D Hamilton

Publications and source records attributed to A D Hamilton.

At least 19 recordsLinked to original sources
PubMed2001

Thermodynamic aspects of dicarboxylate recognition by simple artificial receptors.

Recognition of dicarboxylates by bis-functional hydrogen-bonding receptors displays divergent thermodynamics in different solvent systems. NMR titration and isothermal titration calorimetry indicated that neutral bis-urea and bis-thiourea receptors form exothermic complexes with dicarboxylates in DMSO, with a near zero entropic contribution to binding. The increased binding strength of bis-guanidinium receptors precluded quantitative measurement of binding constants in DMSO, but titration calorimetry offered a qualitative picture of the association. Formation of these 1:1 complexes was also exothermic, but additional endothermic events occurred at both lower and higher host-guest ratios. These events indicated multiple binding equilibria but did not always occur at a discrete 2:1 or 1:2 host-guest molar ratio, suggesting higher aggregates. With increasing amounts of methanol as solvent, bis-guanidinium receptors form more endothermic complexes with dicarboxylates, with a favorable entropy of association. This switch from association driven by enthalpy to one driven by entropy may reflect a change from complexation involving the formation of hydrogen bonds to that promoted by solvent liberation from binding sites.

Binding Sites↗
PubMed2001

Signaling events required for transforming growth factor-beta stimulation of connective tissue growth factor expression by cultured human lung fibroblasts.

It is possible that many of the fibrogenic effects of transforming growth factor-beta (TGF-beta) are mediated by connective tissue growth factor (CTGF). In the present work, we show that TGF-beta1 produces a 5- to 6-fold increase in CTGF expression by cultured human lung fibroblasts that is due mainly to increased transcription. The half-life of CTGF mRNA is 1.96 h, consistent with its role as a cytokine. In addition to requiring Smad activity, based upon the effects of specific inhibitors, the TGF-beta intracellular signaling pathway requires the activity of a phosphatidylcholine-specific phospholipase C, a protein kinase C, and one or more tyrosine kinases. It is also likely that the pathway requires a member of the Ras superfamily of small GTPases, but not trimeric G proteins. Pharmacologic inhibition of TGF-beta stimulation of CTGF expression may be an effective therapeutic approach to a variety of undesirable fibrotic reactions.

Cells, Cultured↗
PubMed2001

Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation.

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

3T3 Cells↗
PubMed2001

Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity.

Malaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are showing deficiencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthesis, and evaluation of potential antimalarial agents against a novel target, protein farnesyltransferase. We show that the most potent compounds are active against Plasmodium falciparum in vitro at submicromolar concentrations.

Alkyl and Aryl Transferases↗
PubMed2001

CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and p27KIP1 expression and apoptosis and inhibits the growth of the human lung adenocarcinoma A-549 in nude mice.

The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH-(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.

Adenocarcinoma↗
PubMed2001

The farnesyltransferase inhibitor, FTI-2153, blocks bipolar spindle formation and chromosome alignment and causes prometaphase accumulation during mitosis of human lung cancer cells.

Even though farnesyltransferase inhibitors (FTIs), a novel class of therapeutic agents presently in clinical trials, have preclinically outstanding anticancer activity and impressive lack of toxicity, their mechanism of action is not well understood. To enhance our understanding of how FTIs inhibit the growth of tumors, we have investigated their effects on cell cycle progression of two human lung cancer cell lines, A-549 and Calu-1. In this report, we show in synchronized A-549 and Calu-1 cells that FTI-2153 treatment resulted in a large accumulation of cells in the mitosis phase of the cell division cycle, with some cells in the G(0)/G(1) phase. Furthermore, microtubule immunostaining and 4,6-diamidino-2-phenylindole DNA staining demonstrated that the FTI-2153-induced accumulation in mitosis is due to the inability of these cells to progress from prophase to metaphase. FTI-2153 inhibited the ability of A-549 and Calu-1 cells to form bipolar spindles and caused formation of monoasteral spindles. Furthermore, FTI-2153 induced a ring-shaped chromosome morphology and inhibited chromosome alignment. Time-lapse videomicroscopy confirmed this result by showing that FTI-2153-treated cells are unable to align their chromosomes at the metaphase plate. FTI-2153 did not affect the localization to the kinetochores of two farnesylated centromeric proteins, CENP-E and CENP-F. Thus, a mechanism by which FTIs inhibit progression through mitosis and tumor growth is by blocking bipolar spindle formation and chromosome alignment.

Alkyl and Aryl Transferases↗
PubMed2001

v-Src transformation is mediated through farnesylated proteins.

Src is an oncoprotein which has been implicated in a number of human malignancies in which it has been shown to be overexpressed and highly activated. The precise mechanism of Src transformation, however, is still poorly understood. We hypothesized that Ras and other farnesylated proteins may mediate Src transformation. To test this hypothesis, v-Src-transfected rat fibroblasts (3Y1) were treated every 72 h with a 15 microM concentration of a farnesyl-transferase inhibitor (FTI). At 2 weeks, a focus formation assay was performed to assess transformation potential. Untreated and FTI-treated v-Src-transfected 3Y1 cells formed a mean of 39 (+/-2.6) and 29.8 (+/-2.9) foci per well, respectively. This 24% decrease was judged to be statistically significant (P = 0.02). Moreover, foci (>90%) in the FTI-treated wells were also consistently smaller than foci in the untreated wells. Western blots with antibody directed toward H-Ras confirmed complete inhibition of Ras farnesylation in the treated cell lines. The specificity of this inhibition was verified by Western blot using antibody specific for Rap1A. The transforming potential of v-Src is inhibited, but not eliminated by FTI treatment. This suggests that v-Src transformation is mediated in part by farnesylated proteins, one of which may be Ras.

Alkyl and Aryl Transferases↗
PubMed2001

Functionalized oligoanthranilamides: modular and conformationally controlled scaffolds.

This paper describes the use of functionalized oligoanthranilamides as conformationally controlled scaffolds for molecular recognition. Oligomers of anthranilamides are stabilized by the formation of intramolecular six-membered hydrogen bonds in a linear strand conformation. Onto alternate anthranilic acid units, we have attached di- or tripeptide recognition units with the potential to form intramolecular hydrogen bonds to an intercalated peptide strand. Using 1H NMR dilution experiments in CDCl(3), we have observed chemical shift changes that are consistent with the formation of an extended hydrogen bonded sheet dimer. We also demonstrate that the bis-alanine functionalized strands are able to form discrete hydrogen bonded complexes with dipeptide substrates and to bind hexanoyl alanylalanine selectively over its benzyl ester. In the presence of excess hydrogen bond donors and acceptors, the oligoanthranilamide strand retained its linear conformation, pointing to the potential of this modular design as a useful and stable scaffold for molecular recognition studies.

Dimerization↗
PubMed2000

Design of growth factor antagonists with antiangiogenic and antitumor properties.

This review describes our recent efforts in the development of novel therapies for cancer. Our primary approach is to design synthetic agents that antagonize the function of growth factors that are critically involved in oncogenesis and angiogenesis. We achieve this by designing synthetic molecules that can recognize the exterior surface of the growth factor and so block the interaction with its receptor tyrosine kinase. A key step is the construction of synthetic agents that contain a large (> 400A2) and functionalized surface area to recognize a complementary surface on the target growth factor. In the course of this work we have discovered a molecule, GFB-111, that binds to PDGF, prevents it from binding to its receptor tyrosine kinase, blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases and DNA synthesis. The binding affinity for PDGF is high (IC50=250 nM) and selective over EGF, IGF-1, aFGF, bFGF and HRGbeta. In nude mouse models GFB-111 also shows significant inhibition of tumor growth and angiogenesis.

Angiogenesis Inhibitors↗
PubMed2000

Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies.

In 1990, more than 10 years after the discovery that the low molecular weight GTPase Ras is a major contributor to human cancer, farnesylation, a lipid posttranslational modification required for the cancer-causing activity of Ras, emerged as a major target for the development of novel anticancer agents. However, it took only 5 years from 1993, when the first farnesyltransferase inhibitors (FTIs) were reported, to 1998 when results from the first phase I clinical trials were described. This rapid progress was due to the demonstration of outstanding antitumor activity and lack of toxicity of FTIs in preclinical models. Although, many FTIs are currently in phase H and at least one is in phase III clinical trial, the mechanism of FTI antitumor activity is not known. In this review a brief summary of the development of FTIs as antitumor agents will be given. The focus of the review will be on important mechanistic and bench-to-bedside translational issues. Among the issues that will be addressed are: evidence for and against inhibition of the prenylation of Ras and RhoB proteins in the mechanism of action of FTIs; implications of the alternative prenylation of K-Ras by geranylgeranyl-transferase I (when FTase is inhibited) in cancer therapy; GGTase I inhibitors (GGTIs) as antitumor agents; effects of FTIs and GGTIs on cell cycle machinery and progression and potential mechanisms by which FTIs and GGTIs induce apoptosis in human cancer cells. A thorough discussion about bench-to-bedside issues relating to hypothesis-driven clinical trials with proof-of-principle in man will also be included. This section will cover issues relating to whether the biochemical target (FTase) is inhibited and the level of inhibition of FTase required for clinical response; are signaling pathways such as H-Ras/PI3K/Akt and/or K-Ras/Raf/MEK/Erk relevant biological readouts?; is Ras (particularly N-Ras and H-Ras) mutation status a good predictor of clinical response?; in phase I trials should effective biological dose, not maximally tolerated dose, be used to determine phase II dose?; and finally, in phase II/III trials what are the most appropriate clinical end points for anti-signaling molecules such as FTIs? Parts of this topic have been recently reviewed (Sebti and Hamilton, 2000c).

Alkyl and Aryl Transferases↗
PubMed2000

Inhibition of farnesyltransferase increases TGFbeta type II receptor expression and enhances the responsiveness of human cancer cells to TGFbeta.

Several small GTPases of the Ras superfamily have been shown to antagonize TGFbeta signaling in human tumor cell lines. Some of these GTPases are post-translationally modified by farnesylation, a lipid modification catalyzed by farnesyltransferase and required for the proteins to attach to membranes and to function. In this study, we investigated the effect of the farnesyltransferase inhibitor FTI-277 on TGFbeta-regulated cell growth and transcription. Treatment of the human pancreatic tumor cell line, Panc-1, with FTI-277 enhanced the ability of TGFbeta to inhibit both anchorage-dependent and -independent tumor cell growth. FTI-277 also enhanced the ability of TGFbeta to induce transcription, as measured by p3TP-lux reporter activity and collagen synthesis. The enhancement of TGFbeta responses by FTI-277 correlated with the stimulation of transcription and protein expression of type II TGFbeta receptor (TbetaRII). Consequently, FTI-277-treated cells exhibited a higher level of TGFbeta binding to its receptor. Thus, inhibition of protein farnesylation stimulates TbetaRII expression, which leads to increased TGFbeta receptor binding and signaling as well as inhibition of tumor cell growth and transformation.

3T3 Cells↗
PubMed2000

RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription.

The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21(WAF) transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchorage-dependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21(WAF) transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.

3T3 Cells↗
PubMed2000

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Alkyl and Aryl Transferases↗
PubMed2000

Nitronate anion recognition and modulation of ambident reactivity by hydrogen-bonding receptors.

Nitronate anions were shown to form complexes in DMSO with hydrogen-bonding receptors such as 1,3-dimethylthiourea 1 (K(a)= 120M(-1)) and bicyclic guanidinium 2 (K(a) = 3200M(-1)). A ditopic bis-thiourea exhibited increased association with substrates, that contained either two nitronates (K(a)= 7000M(-1)) or a combination of nitronate and carboxylate (K(a)=7200M(-1)). Complexation of nitronate resulted in a change in the ambident reactivity during alkylation with p-nitrobenzyl bromide. The predominant reaction pathway was shifted from oxygen alkylation to carbon alkylation as receptor binding strength increased. Kinetic analysis indicated an overall inhibition of nitronate reactivity, and this suggests that greater suppression of the oxygen pathway allows carbon alkylation to predominate.

Alkylation↗
PubMed2000

Protein surface recognition by synthetic receptors based on a tetraphenylporphyrin scaffold.

[structure: see text] Receptors based on a tetraphenylporphyrin scaffold bearing different charged and hydrophobic groups have been synthesized. The interactions of these with horse heart cytochrome c were studied by fluorescence spectroscopy. Receptor 4 was identified to be the strongest synthetic receptor (K(d) = 20 nM) for cytochrome c. The differences in affinity among the receptors reflected a dependence on the number of anionic and hydrophobic groups.

Animals↗