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Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemic

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemic

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemic

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemic

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemic