Synthesis and biological evaluation of a unique heparin mimetic hexasaccharide for structure-activity relationship studies.

To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: role of the cell surface heparan sulfate glycosaminoglycans.

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Genetically engineered mouse melanoma and mouse Lewis lung carcinoma models.

Melanoma B16-F10 cells and Lewis lung carcinoma LL/2 cells were engineered with a bacterial gene -- chloramphenicol acetyl transferase (CAT) -- by establishing stable transductants. Expression of CAT in both cell types did not alter the ability of these cells to grow into tumors when injected subcutaneously into mice. In addition, the measurement of CAT levels in the lung using a simple ELISA assay revealed a close correlation with direct counting of metastatic nodules. Thus, the CAT-expressing cells will likely have wide ranging applications to quantify tumor metastasis especially in situations where visual counting is difficult. The availability of genetically labeled mouse B16-F10 melanoma and Lewis lung carcinoma cell lines will facilitate future studies of the mechanism and progression of cancer and the discovery of new therapies.

Peptide ligands in antibacterial drug discovery: use as inhibitors in target validation and target-based screening.

There is an urgent need to develop novel classes of antibiotics to counter the inexorable rise of resistant bacterial pathogens. Modern antibacterial drug discovery is focused on the identification and validation of novel protein targets that may have a suitable therapeutic index. In combination with assays for function, the advent of microbial genomics has been invaluable in identifying novel antibacterial drug targets. The major challenge in this field is the implementation of methods that validate protein targets leading to the discovery of new chemical entities. Ligand-directed drug discovery has the distinct advantage of having a concurrent analysis of both the importance of a target in the disease process and its amenability to functional modulation by small molecules. VITA is a process that enables a target-based paradigm by using peptide ligands for direct in vitro and in vivo validation of antibacterial targets and the implementation of high-throughput assays to identify novel inhibitory molecules. This process can establish sufficient levels of confidence indicating that the target is relevant to the disease process and inhibition of the target will lead to effective disease treatment.