A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent

A chimeric protein, consisting of streptavidin fused to a cyclic decapeptide with potent inhibitory activity for matrix metalloproteinases (MMP), has been produced in Escherichia coli and purified. The purified chimera formed a tetramer and showed full biotin-binding ability. The chimera was also capable of both binding to MMP-2 and inhibiting its activity. Thus, both the streptavidin moiety and the decapeptide of the chimera are fully functional. This bifunctional nature of the chimera should facilitate the application of the decapeptide since the streptavidin moiety can be used as a specific conjugation site for almost any materials upon biotinylation.     

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.     

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

Natural human monocyte gelatinase and its inhibitor.

Gelatinases produced by stimulated peripheral blood monocytes were detected by substrate zymography and were compared with those derived from tumor cells. Stimulated monocytes were found to produce an 85 kDa gelatinase which co-migrated upon electrophoretic separation and cross-reacted in immunoprecipitation experiments with a phorbol ester inducible metalloprotease from human tumor cells. The intact natural gelatinase (85 kDa), a high molecular weight and complexed gelatinase as well as a proteolytic fragment (25 kDa) were purified by substrate- and antibody-affinity chromatography techniques. Aminoterminal sequence analysis showed that natural monocyte gelatinase occurs as a truncated form of tumor cell gelatinase/type IV collagenase. Furthermore, peripheral blood monocytes were found to also produce a tissue inhibitor of metalloproteases (TIMP). TIMP was co-purified with gelatinase on gelatin sepharose and identified by microsequencing. The balanced and regulated production of gelatinase and TIMP might be important in monocyte migration and tissue remodeling.     

In vivo localization of gelatinases (MMP-2 and -9) by in situ zymography with a selective gelatinase inhibitor

In situ zymography provides a tool to localize proteolytic activity in tissues in vivo. However, it has been difficult to discriminate between the proteases responsible for the detected activity. We used a selective tissue-permeable gelatinase inhibitor, the CTTHWGFTLC-peptide (CTT) in inflamed human gingiva. The CTT-peptide was evidenced to home, target to, and selectively inhibit the areas of gelatinolytic activity in inflamed human gingiva expressing MMP-2 and -9. Gelatinolytic activity, MMP-9 immunoreactivity, and mRNA expression as well as CD-45-positive inflammatory cells colocalized well in the inflamed human gingival connective tissue. Gelatinolytic activity corresponding to MMP-2 colocalized with laminin-5 gamma2-chain immunoreactivity and was detected in the close vicinity of the sulcular basement membrane region. Furthermore, the CTT-peptide inhibited beta-caseinolysis by human MMP-2 and MMP-9 as well as laminin-5 gamma2-chain degradation by MMP-2 in vitro. Thus, the CTT-peptide may prove to be a useful tool (i) to discriminate between gelatinolytic proteases detected by in situ zymography and (ii) to preventMMP-2-dependent induction of epithelial cell migration and gelatinase-dependent tissue destruction in inflammatory and malignant diseases.     

Marcaine, a selective inhibitor of eucaryotic aminoacylation.

The effects of marcaine, a myotoxic drug, on the aminoacylation of transfer ribonucleic acid (rRNA) have been studied. The drug is a potent inhibitor of the acylation of rat liver tRNA with leucine and isoleucine but is only mildly inhibitory (or not inhibitory) to acylation with a number of other amino acids which were tested. Further, marcaine inhibited aminoacylation in cell-free systems using components from several mammalian tissues, including muscle, from yeast, and from wheat germ. No effect of the drug was observed in aminoacylation systems from several bacterial species which were tested. The drug inhibits acylation with leucine and isoleucine competitively but exhibited noncompetitive kinetics when the concentrations of adenosine 5'-triphosphate (ATP) and tRNA were varied. Marcaine was also a competitor of leucine in the ATP--pyrophosphate exchange reaction. Two structural analogues of marcaine, carbocaine and xylocaine, also inhibited acylation of rat liver tRNA with leucine but in a noncompetitive fashion. On a molar basis, marcaine appears to be the most effective inhibitor of the three drugs tested.     

A potent and selective inhibitor targeting human and murine 12/15-LOX

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood–brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC₅₀ potency of 3.4 ± 0.5 μM against h12/15-LOX in vitro and an ex vivo IC₅₀ potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a K_ic of 1.0 ± 0.08 μM and a K_iu of 6.0 ± 3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.     

肿瘤血管靶向药物的研究进展

肿瘤血管在实体瘤的发生发展中具有重要的作用,靶向肿瘤血管的新药研发已成为一个热点领域.抗肿瘤血管的治疗策略分为肿瘤新生血管生成抑制剂（tumor angiogenesis inhibitor,TAI）和肿瘤血管靶向药物（vascular targeting agents,VTAs）两方面的研究.肿瘤新生血管生成抑制剂旨在抑制肿瘤新生血管生成的过程,而肿瘤血管靶向药物则是通过快速而有选择性地破坏肿瘤血管功能,使肿瘤血供受阻,导致肿瘤坏死.VTA类药物分为两类：一是小分子抑制剂（small molecule agents）,利用肿瘤血管和正常组织血管存在的差别选择性地破坏肿瘤血管;另一种是生物制剂（biological agents）,借助能够特异结合肿瘤血管的配体将毒素、凝血诱导剂、凋亡诱导分子等运送到肿瘤血管,引起血管阻塞使肿瘤坏死.     

A short synthesis of argatroban. a potent selective thrombin inhibitor.

Argatroban was synthesized in seven steps from 4-methylpiperidine. The condensation of (+/-)-trans-benzyl 4-methylpipecolic acid ester with N(alpha)-Boc-N(omega)-nitro-L-arginine led to two diastereomers that were separated. One of them is the precursor of argatroban.     

A highly potent and selective inhibitor Roxyl-WL targeting IDO1 promotes immune response against melanoma

Indoleamine 2,3-dioxygenase 1 (IDO1) activity links to immune escape of cancers. Inhibition of IDO1 provides a new approach for cancer treatment. Most clinical IDO1 drugs show marginal efficacy as single agents. On basis of molecular docking and pharmacophore modelling, a novel inhibitor Roxyl-WL was discovered with a half maximal inhibitory concentration (IC50) value of 1?nM against IDO1 and 10–100-fold increased potent activity compared with IDO1 drugs in clinical trials. Roxyl-WL displayed excellent kinase spectrum selectivity with no activity out of the 337 protein kinases. In vitro, Roxyl-WL effectively augmented the proliferation of T cells and reduced the number of regulatory T cell (Tregs).When administered to melanoma (B16F10) tumor-bearing mice orally, Roxyl-WL significantly suppressed tumor growth and induced immune response.     

An oxyanion-hole selective serine protease inhibitor in complex with trypsin.

p-amidinophenylmethylphosphinic acid (AMPA) was designed, synthesized and crystallized in complex with trypsin to study interactions with the oxyanion hole at the S1 site. In comparison to benzamidine, AMPA shows improved activity, which the crystal structure demonstrates to result from hydrogen bonds between the negatively charged phosphinic acid group and the catalytic residues at the oxyanion hole.     

DNA repair inhibition: a selective tumour targeting strategy

Advanced cancer is a leading cause of death in the developed world. Chemotherapy and radiation are the two main treatment modalities currently available. The cytotoxicity of many of these agents is directly related to their propensity to induce DNA damage. However, the ability of cancer cells to recognize this damage and initiate DNA repair is an important mechanism for therapeutic resistance and has a negative impact upon therapeutic efficacy. Pharmacological inhibition of DNA repair, therefore, has the potential to enhance the cytotoxicity of a diverse range of anticancer agents. Moreover, the use of inhibitors of DNA repair or DNA damage signalling pathways appears to provide an exciting opportunity to target the genetic differences that exist between normal and tumour tissue.     

vMIA,a viral inhibitor of apoptosis targeting mitochondria

Human cytomegalovirus encodes a powerful cell death suppressor vMIA (viral mitochondria-localized inhibitor of apoptosis), also known as pUL37x1. vMIA, a product of the immediate early gene UL37 exon 1, is predominantly localized in mitochondria, where it appears to form a complex with adenine nucleotide translocator, believed to be a component of the mitochondrial transition pore complex. vMIA suppresses apoptosis by blocking permeabilization of the mitochondrial outer membrane. Expression of vMIA protects cells against apoptosis triggered by diverse stimuli, including ligation of death receptors, exposure to certain cytotoxic drugs, and infection with an adenovirus mutant deficient in E1B19K. Deletion mutagenesis of vMIA revealed two domains that are necessary and, together, sufficient for its anti-apoptotic activity. The first domain contains a mitochondrial targeting signal. The function of the second domain is still unknown. vMIA does not share any significant amino acid sequence homology with Bcl-2, and, unlike Bcl-2 or Bcl-x(L), it does not bind BAX or VDAC. These structural and functional differences between vMIA and Bcl-2 suggest that vMIA represents a separate class of cell death suppressors. Experiments with vMIA-deficient CMV (human cytomegalovirus) mutants provide strong evidence that the anti-apoptotic function of vMIA is required to prevent CMV-induced apoptosis, and is necessary for viral replication. In addition to vMIA, UL37 encodes two longer splice-variant proteins, gpUL37 and GP37(M). Biological functions of these proteins have not yet been identified, and may be unrelated to their anti-apoptotic activity. The identification of vMIA and the finding that its anti-apoptotic function is required for CMV replication provides a rationale for the development of anti-CMV pharmaceuticals that would inactivate vMIA and thus restore apoptosis in cells infected with CMV.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Knipholone, a selective inhibitor of leukotriene metabolism

Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC(50) value of 4.2microM. However, at a concentration of 10microg/ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50microM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, non-enzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell.     

Toward a PKB inhibitor: modification of a selective PKA inhibitor by rational design

Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.     

Imidazole: a selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 MICRONG/ML; DETERMINED BY BIOASSAY). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PGH2. Several imidazole analogues and other substances were tested but only 1-methyl-imidazole was more potent than imidazole itself. The use of imidazole to inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.