Publications (11)34.93 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.
    No preview · Article · Dec 2008 · Journal of Medical Virology
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    [Show abstract] [Hide abstract] ABSTRACT: In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.
    Full-text · Article · Dec 2008 · Clinical Cancer Research
  • [Show abstract] [Hide abstract] ABSTRACT: In an effort to develop new agents and molecular targets for the treatment of cancer, aspargine-glycine-arginine (NGR)-targeted liposomal doxorubicin (TVT-DOX) is being studied. The NGR peptide on the surface of liposomal doxorubicin (DOX) targets an aminopeptidase N (CD13) isoform, specific to the tumor neovasculature, making it a promising strategy. To further understand the molecular mechanisms of action, we investigated cell binding, kinetics of internalization as well as cytotoxicity of TVT-DOX in vitro. We demonstrate the specific binding of TVT-DOX to CD13-expressing endothelial [human umbilical vein endothelial cells (HUVEC) and Kaposi sarcoma-derived endothelial cells (SLK)] and tumor (fibrosarcoma, HT-1080) cells in vitro. Following binding, the drug was shown to internalize through the endosomal pathway, eventually leading to the localization of doxorubicin in cell nuclei. TVT-DOX showed selective toxicity toward CD13-expressing HUVEC, sparing the CD13-negative colon-cancer cells, HT-29. Additionally, the nontargeted counterpart of TVT-DOX, Caelyx, was less cytotoxic to the CD13-positive HUVECs demonstrating the advantages of NGR targeting in vitro. The antitumor activity of TVT-DOX was tested in nude mice bearing human prostate-cancer xenografts (PC3). A significant growth inhibition (up to 60%) of PC3 tumors in vivo was observed. Reduction of tumor vasculature following treatment with TVT-DOX was also apparent. We further compared the efficacies of TVT-DOX and free doxorubicin in the DOX-resistant colon-cancer model, HCT-116, and observed the more pronounced antitumor effects of the TVT-DOX formulation over free DOX. The potential utility of TVT-DOX in a variety of vascularized solid tumors is promising.
    No preview · Article · Dec 2007 · Anti-Cancer Drugs
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    [Show abstract] [Hide abstract] ABSTRACT: Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki, ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.
    Full-text · Article · Dec 2007 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: A series of lysine sulfonamide analogues bearing Nε-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.
    No preview · Article · Sep 2006 · ChemInform
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    [Show abstract] [Hide abstract] ABSTRACT: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.
    Full-text · Article · Aug 2006 · Biochemical and Biophysical Research Communications
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    [Show abstract] [Hide abstract] ABSTRACT: A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.
    Preview · Article · Aug 2006 · Bioorganic & Medicinal Chemistry Letters
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    [Show abstract] [Hide abstract] ABSTRACT: We have previously observed that the synthetic peptide corresponding to amino acids 31-45 (PCK3145) of PSP94 can reduce prostate tumor growth in vivo. Moreover, a recently concluded phase IIa clinical trial with patients with hormone refractory prostate cancer indicated that PCK3145 down-regulates the levels of plasma matrix metalloproteinase (MMP)-9, a MMP involved in metastasis and tumor angiogenesis. The purpose of our study was to investigate the molecular mechanisms of action of PCK3145 and whether this peptide could antagonize tumor neovascularization. We show that, in a syngeneic in vivo model of rat prostate cancer, the expression of endothelial cell (EC) specific CD31, a marker of tumor vessel density, was decreased by 43% in PCK3145-treated animals. In vitro, PCK3145 specifically antagonized in a dose-dependent manner the VEGF-induced ERK phosphorylation as well as the phosphorylation of the VEGFR-2 in cultured EC (HUVEC). These anti-VEGF effects were partly reproduced by pharmacological inhibitors such as PD98059 and PTK787, suggesting that PCK3145 inhibits the tyrosine kinase activity associated to VEGFR-2, which in turn prevents intracellular signalling through the MAPK cascade. Moreover, PCK3145 was also found to inhibit the PDGF-induced phosphorylation of PDGFR in smooth muscle cells. Finally, PCK3145 inhibited in vitro EC tubulogenesis and VEGF-induced MMP-2 secretion suggesting its potential implication as an antiangiogenic agent. Our study demonstrates that PCK3145 interferes with the tyrosine kinase activity associated with VEGF signalling axis in EC. The antiangiogenic properties of this peptide could be highly beneficial and exploited in novel antiangiogenic therapies, for patients with various cancers.
    Full-text · Article · May 2006 · International Journal of Cancer
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    [Show abstract] [Hide abstract] ABSTRACT: PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94 which can reduce experimental skeletal metastases and prostate tumor growth. These anti-metastatic and anti-tumoral effects of PCK3145 are partially explained by the in-vivo and in-vitro decrease in matrix metalloproteinase (MMP)-9 extracellular levels through as yet unidentified molecular mechanisms of action. Gelatin zymography and immunoblots were used to monitor the levels of secreted MMP-9 from HT-1080 cells. Flow cytometry was used to monitor HT-1080 cell surface binding of FITC-labeled PCK3145 and biotin-labeled laminin. PCK3145-coated cell culture dishes were used to monitor cell adhesion. HT-1080 cell lysates were used for immunoblotting of HuR, extracellular signal-regulated protein kinase (ERK) and phospho-ERK. Total RNA was isolated and RT-PCR used to monitor HuR gene expression. We found that PCK3145 bound to the HT-1080 cell surface and that this binding rapidly triggered ERK phosphorylation that, ultimately, led to a reduction of secreted MMP-9. Laminin inhibited both cell surface binding and ERK phosphorylation by PCK3145. Overexpression of the 67-kDa laminin receptor led to an increased binding of the cells to PCK3145. HuR, a protein that can bind to and stabilize MMP-9 mRNA, was found to be downregulated by PCK3145. The mitogen-activated protein kinase/ERK (MEK) inhibitor PD98059 as well as native laminin and SIKVAV laminin-derived peptide prevented that downregulation. Our data suggest that PCK3145 rapidly triggers intracellular signaling through cell surface laminin receptors. This leads to decreased HuR expression and subsequent destabilization of MMP-9 transcripts. This is the first molecular evidence demonstrating the intracellular signaling and anti-metastatic mechanism of action of PCK3145 that leads to the inhibition of MMP-9 secretion.
    Full-text · Article · May 2006 · Anti-Cancer Drugs
  • [Show abstract] [Hide abstract] ABSTRACT: This review focuses on the promising roles of prostate secretory protein of 94 amino acids (PSP-94) and one of its derived peptides (PCK3145) as potential therapeutic modalities for prostate cancer and its associated complications. Evaluation of these compounds was carried out in vitro and in vivo using syngeneic models of rat prostate cancer. Overproduction of parathyroid hormone-related protein (PTHrP) results in the development of hypercalcemia of malignancy in several malignancies including prostate cancer. In order to evaluate the effect of PSP-94 and PCK3145 on prostate cancer progression, the rat Dunning R3227 MatLyLu cell line transfected with full-length cDNA encoding PTHrP (MatLyLu-PTHrP) was used. As the main pathogenetic factor of hypercalcemia of malignancy, overexpression of PTHrP was aimed at mimicking the hypercalcemic nature seen in patients suffering from late-stage cancer. In vitro studies showed that PSP-94 and PCK3145 can cause a dose-dependent inhibition in the growth of MatLyLu-PTHrP cells. For in vivo studies, male Copenhagen rats were inoculated either s.c. into the right flank or directly into the left ventricle via intracardiac (i.c.) inoculation with MatLyLu-PTHrP cells. In these models, s.c. injection of MatLyLu cells results in the development of primary tumor growth, whereas i.c. inoculation routinely results in the development of experimental skeletal metastases in the lumbar vertebrae causing hind-limb paralysis. Administration of PSP-94 and PCK3145 into tumor-bearing animals resulted in a dose-dependent inhibition of primary tumor growth, and tumoral and plasma PTHrP levels, and in the reduction of plasma calcium levels. Additionally, treatment with PSP-94 or PCK3145 caused an inhibition of skeletal metastases resulting in a significant delay in the development of hind-limb paralysis. Interestingly, equimolar concentrations of PCK3145 were shown to be more effective in delaying the development of experimental skeletal metastases as compared to PSP-94. One of the possible mechanisms of action of these modalities is through the induction of apoptosis which was observed by both in-vitro and in-vivo analyses of MatLyLu-PTHrP cells and tumors. Several intracellular mechanisms can also be involved in inhibiting PTHrP production and anti-tumor effects of PSP-94 and PCK3145. Collectively, these studies warrant the continued clinical development of these agents as therapeutic agents for patients with hormone-refractory prostate cancer.
    No preview · Article · Dec 2005 · Anti-Cancer Drugs
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    [Show abstract] [Hide abstract] ABSTRACT: PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.
    Full-text · Article · Feb 2005 · Clinical and Experimental Metastasis