An orally delivered small-molecule formulation with antiangiogenic and anticancer activity

Targeting angiogenesis, the formation of blood vessels, is an important modality for cancer therapy. TNP-470, a fumagillin analog, is among the most potent and broad-spectrum angiogenesis inhibitors. However, a major clinical limitation is its poor oral availability and short half-life, necessitating frequent, continuous parenteral administration. We have addressed these issues and report an oral formulation of TNP-470, named Lodamin. TNP-470 was conjugated to monomethoxy-polyethylene glycol-polylactic acid to form nanopolymeric micelles. This conjugate can be absorbed by the intestine and selectively accumulates in tumors. Lodamin significantly inhibits tumor growth, without causing neurological impairment in tumor-bearing mice. Using the oral route of administration, it first reaches the liver, making it especially efficient in preventing the development of liver metastasis in mice. We show that Lodamin is an oral nontoxic antiangiogenic drug that can be chronically administered for cancer therapy or metastasis prevention.     

Design,synthesis, biological evaluation and dynamics simulation of indazole derivatives with antiangiogenic and antiproliferative anticancer activity

VEGFR-2 has a pivotal role in promoting cancer angiogenesis. Herein, two series of novel indazole-based derivatives were designed, synthesized and evaluated for their in vitro inhibitory action against VEGFR-2 kinase enzyme. The second series 11a-e exhibited better potency than the first one 7a-d and 8a-f. Compounds 11b, 11c and 11e exhibited the most potent action, with IC₅₀ of 5.4 nM, 5.6 nM and 7 nM, respectively. As a measure of cellular VEGFR-2 inhibition, compounds 11b and 11c showed strong inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 80% and 99.6% inhibition at 10 μM concentration, respectively. Attempting to interpret SAR of the synthesized compounds, and provide a basis for further optimization; a comprehensive modeling study was implemented. Molecular docking, dynamics simulation and free energy calculation of the synthesized compounds along with known VEGFR-2 inhibitors were applied. The study illustrated the effect of several factors on VEGFR-2 inhibition, such as the interaction with solvent accessible region of the enzyme, the presence of NH linker and the degree of conformational restriction. Finally, our compounds were evaluated for their in vitro anti-proliferative effect against the full NCI panel of cancer cell lines, where compounds 11a and 11c displayed mean GI% of 93 and 130%, respectively, and showed partly a better behavior than the FDA approved drug sorafenib, with respect to activity (GI₅₀) and safety (LC₅₀) against several cell lines. Thus, compound 11c represents a promising candidate for cancer treatment through antiangiogenic dependent and antiangiogenic independent modes of action.     

Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGbeta (IC50 values > 100 microM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 microM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor-binding molecules with potent anticancer and antiangiogenic activity.     

A new phage-display tumor-homing peptide fused to antiangiogenic peptide generates a novel bioactive molecule with antimelanoma activity

Phage-display peptide libraries have been widely used to identify specific peptides targeting in vivo tumor cells and the tumor vasculature and playing an important role in the discovery of antitumor bioactive peptides. In the present work, we identified a new melanoma-homing peptide, (-CVNHPAFAC-), using a C7C phage-display library directed to the developing tumor in syngeneic mice. Phage were able to preferentially target melanoma in vivo, with an affinity about 50-fold greater than that with normal tissue, and the respective synthesized peptide displaced the corresponding phage from the tumor. A preferential binding to endothelial cells rather than to melanoma cells was seen in cell ELISA, suggesting that the peptide is directed to the melanoma vasculature. Furthermore, the peptide was able to bind to human sonic hedgehog, a protein involved in the development of many types of human cancers. Using a new peptide approach therapy, we coupled the cyclic peptide to another peptide, HTMYYHHYQHHL-NH(2), a known antagonist of VEGFR-2 receptor, using the GYG linker. The full peptide CVNHPAFACGYGHTMYYHHYQHHL-NH(2) was effective in delaying tumor growth (P < 0.05) and increasing animal survival when injected systemically, whereas a scramble-homing peptide containing the same antagonist did not have any effect. This is the first report on the synthesis of a tumor-homing peptide coupled to antiangiogenic peptide as a new anticancer therapeutics.     

NMR solution structure of the angiostatic peptide anginex

Anginex, a designed peptide 33mer, is known to function both as an antiangiogenic and bactericidal agent. Solving the NMR solution structure of the peptide is key to understand better its structure-activity relationships and to design more bioactive peptides and peptide mimetics. However, structure elucidation of anginex has been elusive due to subunit exchange-induced resonance broadening. Here, we found that performing NMR structural studies in a micellar environment abolishes exchange broadening and allows the structure of anginex to be determined. Anginex folds in an amphipathic, three-stranded antiparallel beta-sheet conformation with functionally key hydrophobic residues lying on one face of the beta-sheet and positively charged, mostly lysine residues, lying on the opposite face. Structural comparison is made with a homologous, yet relatively inactive peptide, betapep-28. These results contribute to the design of peptidomimetics of anginex for therapeutic use against angiogenically-related diseases like cancer, as well as infectious diseases.     

Anti-angiogenesis and anti-tumor activity of recombinant anginex

Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment.     

Design and synthesis of a beta-amino phosphotyrosyl mimetic suitably protected for peptide synthesis

Mimetics of phosphotyrosine (pTyr) such as phosphonomethylphenylalanine (Pmp) have traditionally retained alpha-amino functionality. However, beta-amino acids represent isomeric variants, which may exhibit properties that are distinct from the parent. Reported herein is the first beta-amino pTyr mimetic (Pmp(beta)) bearing protection suitable for peptide synthesis. Preparation of Pmp(beta) was accomplished enantioselectively in 43% overall yield from commercially available 4-vinylbenzyl chloride.     

Purification and partial biologic characterization of a human lymphocyte-derived peptide with potent neutrophil-stimulating activity

A novel neutrophil-activating peptide is detected in supernatants from mitogen-stimulated human T lymphocyte preparations. This chemotaxin was purified to apparent homogeneity by sequential Gel G-75 permeation chromatography, wide pore reversed phase (RP-8) HPLC, size exclusion HPLC, and reversed phase (RP-18) HPLC. Additional characterization of this lymphocyte-derived neutrophil-activating peptide (LYNAP) resulted in a single peak upon reversed phase HPLC and size exclusion HPLC. SDS-PAGE under nonreducing conditions revealed a single line at 10 kDa. LYNAP stimulated neutrophil chemotaxis (ED50 of 3 +/- 3 ng/ml), chemokinesis (ED50 of 2 +/- 2 ng/ml), and caused degranulation of cytochalasin B pretreated human neutrophils (ED50 of 20 ng/ml). In purified human monocytes, chemotactic responses to LYNAP at doses up to 100 ng/ml were absent, indicating nonidentity with a lymphocyte-derived monocyte chemotactic factor previously described by other workers. LYNAP shows biochemical and biologic similarities to a recently detected monocyte-derived neutrophil-activating peptide (MONAP). Moreover, desensitization experiments revealed cross-deactivation between LYNAP and MONAP, not, however, between these two chemotactic peptides and other well characterized polymorphonuclear leukocyte chemotaxins, e.g., C5a, FMLP, leukotriene B4, or platelet-activating factor. This finding points toward structure identity or homology of both chemotaxins, MONAP and LYNAP.     

A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library

Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 microm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel was completely inhibited in the presence of 10 microm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and beta-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-beta-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure.     

Enhanced anticancer activity of gemcitabine in combination with noscapine via antiangiogenic and apoptotic pathway against non-small cell lung cancer

Background

The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos) and Gemcitabine (Gem) combination (NGC) against non-small cell lung cancer (NSCLC) and to elucidate the underlying mechanism of action.

Methods

Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues.

Results

The CI values (<0.59) were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups.

Conclusion

Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer.     

Biological evaluation of tubulysin A: a potential anticancer and antiangiogenic natural product

Tubulysin A (tubA) is a natural product isolated from a strain of myxobacteria that has been shown to depolymerize microtubules and induce mitotic arrest. The potential of tubA as an anticancer and antiangiogenic agent is explored in the present study. tubA shows potent antiproliferative activity in a panel of human cancer cell lines irrespective of their multidrug resistance properties. It induces apoptosis in cancer cells but not in normal cells and shows significant potential antiangiogenic properties in several in vitro assays. It is efficacious in initial animal studies using a hollow fibre assay with 12 different human tumour cell lines. This study suggests that both in vitro and preclinical profiles of tubA may translate into clinically useful anticancer properties.     

Design, synthesis and characterisation of a peptide with oxaloacetate decarboxylase activity

The synthesis of Oxaldie-3, a synthetic 31-residue peptide with oxaloacetate decarboxylase activity, is described. Biophysical characterisation by gel filtration, CD and NMR spectroscopy indicated that the peptide adopted a folded structure in solution. Oxaldie-3 was an efficient catalyst at concentrations as low as 2 microM, 100-fold lower than the previously described Oxaldie-2, which relied on aggregating alpha-helices for activity. Oxaldie-3 speeded decarboxylation by more than three orders of magnitude relative to simple amines.     

Template-constrained cyclic peptide analogues of somatostatin: subtype-selective binding to somatostatin receptors and antiangiogenic activity

Beta-turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological activities, contains a beta-turn in its bioactive conformation. The beta-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a beta-turn conformation, as measured by somatostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr-D-Trp-Lys-Val-tetrapeptide were linked by scaffolds based on either an N,N'-dimethyl-N,N'-diphenylurea or a substituted biphenyl system (DJS631 and DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with affinities as high as 1 nM. DJS811, at a dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of structured turn scaffolds allows beta-turn sequences to be contained in the context of a compact structure, with less peptidic nature and potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of beta-turn formation, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.     

Time-dependent inhibition of protein farnesyltransferase by a benzodiazepine peptide mimetic

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the prenylation of proteins with a carboxy-terminal tetrapeptide sequence called a CaaX box, where C refers to cysteine, "a" refers to an aliphatic residue, and X typically refers to methionine, serine, or glutamine (FTase), or to leucine (GGTase-I). Marsters and co-workers [(1994) Bioorg. Med. Chem. 2, 949--957] developed inhibitors of FTase with cysteine and methionine attached to an inner hydrophobic benzodiazepine scaffold. We found that the most potent of these compounds (BZA-2B) resulted in the time-dependent inhibition of FTase. The K(i) of BZA-2B for FTase, which is the dissociation constant of the initial complex, was 79 +/- 13 nM, and the K(i)*, which is the overall dissociation of inhibitor for all enzyme forms, was 0.91 +/- 0.12 nM. The first-order rate constant for the conversion of the initial complex to the final complex was 1.4 +/- 0.2 min(-1), and that for the reverse process was 0.016 +/- 0.002 min(-1). The latter rate constant corresponds to a half-life of the final complex of 45 min. Our experiments favor the notion that the inhibitor binds to the FTase--farnesyl diphosphate complex which then undergoes an isomerization to form a tighter FTase*--farnesyl diphosphate--BZA2-B complex. Diazepam, a compound with a benzodiazepine nucleus but lacking amino acid extensions, was a weak (K(i) = 870 microM) but not time-dependent inhibitor of FTase. Cys-Val-Phe-Met and Cys-4-aminobenzoyl-Met were instantaneous and not time-dependent inhibitors of FTase. Furthermore, BZA-4B, with a leucine specificity determinant, was a classical competitive inhibitor of GGTase-I and not a time-dependent inhibitor.     

Synthesis and antiaggregative activity of a new RGDF mimetic

m-[4-Oxo-4-(piperazin-1-yl)butyrylamino)]benzoyl-D,L-beta-(3,4-methylenedioxyphenyl)-beta-alanine, a new mimetic of the peptide RGDF, was synthesized. This compound inhibited ADP-induced platelet aggregation in human blood plasma enriched with platelets with IC50 3.5 x 10(-8) M. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.     

An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

Lecithin cholesterol acyltransferase (LCAT) plays a key role in the reverse cholesterol transport (RCT) process by converting cholesterol to cholesteryl ester to form mature HDL particles, which in turn deliver cholesterol back to the liver for excretion and catabolism. HDL levels in human plasma are negatively correlated with cardiovascular risk and HDL functions are believed to be more important in atheroprotection. This study investigates whether and how D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, influences LCAT activity in the completion of the RCT process. We demonstrated that the apparent rate constant value of the LCAT enzyme reaction gives a measure of LCAT activity and determined the effects of free metals and a reducing agent on LCAT activity, showing an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ and no inhibition with β-mercaptoethanol up to 10 mM. We reconstituted nano-disc particles using apoA-I or D-4F with phospholipids. These particles elicited good activity in vitro in the stimulation of cholesterol efflux from macrophages through the ATP-binding cassette transporter A1 (ABCA1). With these particles we studied the LCAT activity and demonstrated that D-4F did not activate LCAT in vitro. Furthermore, we have done in vivo experiments with apoE-null mice and demonstrated that D-4F (20 mg/kg body weight, once daily subcutaneously) increased LCAT activity and HDL level as well as apoA-I concentration at 72 hours post initial dosing. Finally, we have established a correlation between HDL concentration and LCAT activity in the D-4F treated mice.     

Structural refinement of protein A mimetic peptide

A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.     

p22 is a novel plasminogen fragment with antiangiogenic activity.

Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.