Stephen Swenson

Anti-Angiogenesis and RGD-Containing Snake Venom Disintegrins

Angiogenesis is the fundamental process by which new blood vessels are formed. Extensive research has shown that this event can be co-opted by tumors to ensure their growth, survival and metastasis. The study of tumor angiogenesis therefore represents a promising area of research for development of anti-cancer therapeutics. Integrins, a family of cell surface molecules, are a major target of interest as they are known to play a vital role in pathological angiogenesis. Remarkably, small disulfide-rich peptides known as disintegrins, isolated from the venoms of various snake species have been found to bind integrins with extremely high affinity and block their function. Disintegrins are capable of inhibiting several aspects of tumor cell behavior both in vitro and in vivo, including adhesion, migration, invasion, metastasis and angiogenesis. In this review, we will briefly discuss tumor angiogenesis and molecules implicated in the angiogenic process, with a special focus on the role of integrins. We will also discuss therapeutic approaches towards the treatment of tumor angiogenesis, including non-integrin-targeted agents currently in clinical trials. We will summarize the major findings from studies using disintegrins to target integrin-associated angiogenesis in cancer models. Finally, we will present results obtained in our laboratory using the novel dimeric disintegrin, contortrostatin (CN), in studies of endothelial cells and models of breast, ovarian and prostate cancer. In summary, disintegrins represent an exciting new class of molecules that can potentially be used in a clinical setting to inhibit angiogenesis and augment conventional chemotherapeutic agents in the treatment of cancer.

Development of a Novel Recombinant Disintegrin, Contortrostatin, as an Effective Anti-Tumor and Anti-Angiogenic Agent

Contortrostatin (CN) (Mr 13,500 Da) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each chain), which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. In previous studies, we have shown that native CN administered in a liposomal formulation exhibits potent anti-angiogenic and tumor growth inhibitory activities. Current isolation of the protein from crude venom is difficult and prohibitively expensive for translation into the clinic. In this report, we describe a method amenable to large-scale production of a soluble monomeric form of recombinant CN with biologic activity; the protein is expressed directly in the cytoplasm of an engineered bacterial system with an expression yield of approximately 20 mg/l of culture. We present here the in vitro assays as well as the anti-tumor and anti-angiogenic evaluation of liposomal recombinant CN in an orthotopic, xenograft model of human breast cancer.

Contortrostatin, a Snake Venom Disintegrin with Anti-Angiogenic and Anti-Tumor Activity

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins αvβ3, αvβ5, and/or α5β1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.

The Disintegrin Contortrostatin in Combination With Docetaxel Is a Potent Inhibitor of Prostate Cancer In Vitro and In Vivo

There are few available treatments for hormone refractory prostate cancer. Through the inhibition of integrins, contortrostatin (CN) effects tumor cell growth directly as well as through the inhibition of angiogenesis. The effect of CN in combination with docetaxel on prostate cancer cell lines in vitro and in vivo is evaluated in the present study.

CHIMERIC FIBROLASE: COVALENT ATTACHMENT OF AN RGD-LIKE PEPTIDE TO CREATE A POTENTIALLY MORE EFFECTIVE THROMBOLYTIC AGENT

We have prepared an agent possessing both thrombolytic and antiplatelet properties, by conjugating fibrolase, a direct-acting fibrinolytic enzyme isolated from southern copperhead venom, to a peptide which inhibits platelet aggregation. Heterobifunctional coupling reagents, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)-toluamido]hexanoate (Sulfo-LC-SMPT), were used in a molar ratio of 10:1 (coupling agent/fibrolase). The N-hydroxy-succinimide of the coupling agent reacts with surface epsilon-amino groups of lysine residues on fibrolase and provides a dithio group that is highly reactive with small thiol compounds. The derivatives obtained in the first reaction contain approximately two moles of 2-pyridyl disulphide per mole of enzyme. These derivatives were then reacted with the free thiol group in an antiplatelet peptide at a molar ratio of 2:1 (peptide/fibrolase). The peptide-fibrolase conjugate was purified by cation exchange HPLC and analyzed by amino acid analysis. The conjugate contains one mole peptide per mole of fibrolase and retains approximately 85% fibrinolytic activity. The IC50 for inhibition of platelet aggregation in human PRP is 300 nM for the conjugate and 67 nM for the antiplatelet peptide. These results demonstrate the successful formation of a novel chimeric protein with bifunctional activity.

Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with ECs ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

Development of a chimeric recombinant disintegrin as a cost-effective anti-cancer agent with promising translational potential.

Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) Escherichia coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200 mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results that can be partially explained by potent tumor apoptotic effects.

Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 Å resolution

Two acostatin heterodimers interact together to form an αββα tetramer.

Purification, crystallization and preliminary X-ray analysis of the disintegrin contortrostatin from Agkistrodon contortrix contortrix snake venom

Disintegrins are cysteine-rich RGD-containing peptides that block tumor-cell implantation and angiogenesis. Contortrostatin, a homodimeric disintegrin (64 residues in each chain) from southern copperhead snake venom, has been purified to homogeneity and crystallized. Initial attempts at crystallization led to a form grown from polyethylene glycol (PEG), which crystallizes in the orthorhombic space group C222(1), with unit-cell parameters a = 57.39, b = 139.55, c = 78.98 A, and diffracts to a resolution limit of 3.2 A. Very recently, a new crystalline form of the title protein has been obtained grown from ammonium sulfate [(NH(4))(2)SO(4)] as a precipitant having a space group of P2(1)2(1)2(1), with unit-cell parameters a = 37.52, b = 59.93, c = 121.37 A. These improved crystals diffract to a resolution limit of 1.7 A.

Contortrostatin, a homodimeric disintegrin isolated from snake venom inhibits herpes simplex virus entry and cell fusion.

BACKGROUND Herpes simplex virus (HSV) causes significant health problems from periodical skin and corneal lesions to encephalitis. HSV entry provides a unique opportunity for therapeutic intervention. In this study, we evaluated contortrostatin (CN), an Arg-Gly-Asp motif containing disintegrin isolated from snake venom, as a novel therapeutic agent with ability to block HSV entry and related membrane fusion. METHODS In vitro efficacy of CN against HSV was determined using an HSV type-1 (HSV-1) entry assay based on the measurement of β-galactosidase reporter activity originating from the genome of a recombinant strain of HSV-1(KOS) gL86. HSV-1 glycoprotein-mediated cell-to-cell fusion was used to study the effect of CN on polykaryocyte formation. Primary as well as transformed cell lines were used for this study. RESULTS Pre-treatment of Chinese hamster ovary (CHO-K1) cells expressing HSV-1 glycoprotein D receptors and primary cultures of human corneal fibroblasts (CF) with CN resulted in the inhibition of entry, cell-to-cell fusion, and polykaryocyte formation. Interestingly, a more pronounced anti-HSV-1 effect was observed in naturally susceptible CF than CHO-K1 cells. CONCLUSIONS CN, a novel venom-derived peptide, exhibits the ability to block two key steps, entry and cell-to-cell fusion, in HSV infection. Showing strong promise for development as an anti-HSV agent, it also demonstrates better prophylactic efficacy in primary cells.