The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in hum... more The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in human hepatocellular carcinoma (HCC) and is associated with poor survival in HCC patients. Compounds that inhibit the DPP4 enzyme family, such as talabostat and ARI-4175, can mediate tumour regression by immune-mediated mechanisms that are believed to include NLRP1 activation. This study investigated the expression and activity of the DPP4 family during the development of HCC and evaluated the efficacy of ARI-4175 in the treatment of early HCC in mice. This first report on this enzyme family in HCC-bearing mice showed DPP9 upregulation in HCC, whereas intrahepatic DPP8/9 and DPP4 enzyme activity levels decreased with age. We demonstrated that ARI-4175 significantly lowered the total number of macroscopic liver nodules in these mice. In addition, ARI-4175 increased intrahepatic inflammatory cell infiltration, including CD8+ T cell numbers, into the HCC-bearing livers. Furthermore, ARI-4175 a...
Background: Class A amphipathic helical peptide analogs of apo-AI mimetic peptides are potential ... more Background: Class A amphipathic helical peptide analogs of apo-AI mimetic peptides are potential emerging therapeutic approaches to improve HDL function to decrease atherosclerosis. Using retro-inversal sequence approach to synthesize Reverse (Rev) D-4F apo-AI mimetic peptide with D-amino acids and reverse order to maintain exact alignment and superimposable to the parent L-4F peptide, we have recently shown that Rev D-4F peptide significantly reduces aortic root atherosclerosis in apo E-null mice without affecting plasma total and HDL-cholesterol (Qin, et al. Circulation 2005, 112 (17): II-110). In order to define the mechanisms, using in-vivo and in-vitro studies we investigated the effect of Rev D-4F on aortic lesion macrophage content in apo E-null mice, and human aortic endothelial cell (HAEC) LDL oxidation, VCAM-1 expression, and monocyte adhesion, key pathobiological processes involved in atherogenesis. Methods: Three groups of 4 week old apoE-null mice were fed chow diet, and administered water (control), Rev-D4F, or L-4F mimetic peptides (1.6 mg/day, n=6/group) orally in drinking water for 6 weeks. Lesion macrophage content was measured by immunohistochemical staining using monoclonal anti-mouse MOMA2 antibody as a specific marker for monocyte/macrophage in aortic root sections. HAEC LDL oxidation, VCAM-1 expression, and adhesion assays were done by measuring thiobarbituric acid reactive substances (TBARS), Real-time PCR analysis, ELISA, and adhesion rate using fluorescently labeled monocytes respectively. Results and Conclusions: Rev-D4F significantly (p=0.0009) reduced macrophage content in lesions by 47% compared to controls. L-4F mimetic peptide had no effect on macrophage content. In-vitro studies indicated that Rev D-4F significantly inhibited endothelial cell LDL oxidation, TNF-α- and LPS-induced VCAM-1, and oxidized LDL-induced monocyte adhesion. The data suggest that Rev-D4F, through inhibiting inflammatory and LDL oxidative events, reduces endothelial monocyte migration, lesion macrophage content and atherosclerosis.
In the past few decades, the pre‐stressed anchor system has been extensively used to increase the... more In the past few decades, the pre‐stressed anchor system has been extensively used to increase the stability of slope among the Taiwan Island. Several disasters have occurred that caused by the sudden failure of pre‐stressed anchor system. This article presents a real‐time ...
Journal of cardiovascular pharmacology and therapeutics, 2012
Synthetic class A amphipathic helical peptide analogs of apolipoprotein-AI (apoAI; with varied ph... more Synthetic class A amphipathic helical peptide analogs of apolipoprotein-AI (apoAI; with varied phenylalanine residues) are emerging therapeutic approaches under investigation for atherosclerosis. Utilizing retroinverso sequencing, we designed reverse-D4F (Rev-D4F) peptide with 18 d-amino acids containing 4 phenylalanine residues and reverse order that allows the side chain residues to be of exact alignment and superimposable to those of the parent l-amino acid peptide. This study examined the effect of Rev-D4F on atherosclerosis in apolipoprotein E (apoE)-null mice and the underlying mechanisms. ApoE-null mice were fed a chow diet and administered water (control), Rev-D4F, or L4F mimetic peptides (0.4 mg/mL, equivalent to 1.6 mg/d) orally in drinking water for 6 weeks. Aortic root atherosclerotic lesion area, lesion macrophage content, and the ability of plasma high-density lipoprotein (HDL) to influence monocyte chemotaxis were measured. Rev-D4F significantly decreased aortic sinus...
The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal ce... more The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20‐ and 1.3‐fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and i...
Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably e... more Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity.
Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It ha... more Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It has antihyperglycemic activity from inhibition of DPPIV but also striking anticancer activity and a toxicity for which the mechanisms are unknown. 1 cyclizes at physiological pH, which attenuates its inhibitory potency >100-fold, which is a "soft drug" effect. Here we show that this phenomenon can be exploited to create prodrugs with unique properties and potential for selective in vivo targeting. Enzyme-mediated release delivers 1 to the target in the active form at physiological pH; cyclization attenuates systemic pharmacological effects from subsequent diffusion. This "pro-soft" design is demonstrated with a construct activated by and targeted to DPPIV, including in vivo results showing improved antihyperglycemic activity and reduced toxicity relative to 1. Pro-soft derivatives of 1 can help to illuminate the mechanisms underlying the three biological activities, or to help localize 1 at a tumor and thereby lead to improved anticancer agents with reduced toxicity. The design concept can also be applied to a variety of other boronic acid inhibitors.
Efficient syntheses of 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine and 4‐carboxy‐l‐phenylalani... more Efficient syntheses of 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine and 4‐carboxy‐l‐phenylalanine within the context of the pentapeptide Ac‐Ile‐X‐Gly‐Glu‐Phe‐NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide‐based tyrosine kinase inhibitors. The key intermediate, Ac‐Ile‐Phe(4‐formyl)‐Gly‐Glu(O‐tBu)‐Phe‐NH2, was synthesized by a facile palladium‐catalyzed carbonylation of Ac‐Ile‐Phe(4‐iodo)‐Gly‐Glu(O‐tBu)‐Phe‐NH2. Oxidation of Ac‐Ile‐Phe(4‐formyl)‐Gly‐Glu(O‐tBu)‐Phe‐NH2 with tetrabutylammonium permanganate or addition of di‐t‐butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4‐carboxy‐l‐phenylalanine or 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c‐src than the corresponding pentapeptide wherei...
The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in hum... more The mRNA expression of the dipeptidyl peptidase 4 (DPP4) gene family is highly upregulated in human hepatocellular carcinoma (HCC) and is associated with poor survival in HCC patients. Compounds that inhibit the DPP4 enzyme family, such as talabostat and ARI-4175, can mediate tumour regression by immune-mediated mechanisms that are believed to include NLRP1 activation. This study investigated the expression and activity of the DPP4 family during the development of HCC and evaluated the efficacy of ARI-4175 in the treatment of early HCC in mice. This first report on this enzyme family in HCC-bearing mice showed DPP9 upregulation in HCC, whereas intrahepatic DPP8/9 and DPP4 enzyme activity levels decreased with age. We demonstrated that ARI-4175 significantly lowered the total number of macroscopic liver nodules in these mice. In addition, ARI-4175 increased intrahepatic inflammatory cell infiltration, including CD8+ T cell numbers, into the HCC-bearing livers. Furthermore, ARI-4175 a...
Background: Class A amphipathic helical peptide analogs of apo-AI mimetic peptides are potential ... more Background: Class A amphipathic helical peptide analogs of apo-AI mimetic peptides are potential emerging therapeutic approaches to improve HDL function to decrease atherosclerosis. Using retro-inversal sequence approach to synthesize Reverse (Rev) D-4F apo-AI mimetic peptide with D-amino acids and reverse order to maintain exact alignment and superimposable to the parent L-4F peptide, we have recently shown that Rev D-4F peptide significantly reduces aortic root atherosclerosis in apo E-null mice without affecting plasma total and HDL-cholesterol (Qin, et al. Circulation 2005, 112 (17): II-110). In order to define the mechanisms, using in-vivo and in-vitro studies we investigated the effect of Rev D-4F on aortic lesion macrophage content in apo E-null mice, and human aortic endothelial cell (HAEC) LDL oxidation, VCAM-1 expression, and monocyte adhesion, key pathobiological processes involved in atherogenesis. Methods: Three groups of 4 week old apoE-null mice were fed chow diet, and administered water (control), Rev-D4F, or L-4F mimetic peptides (1.6 mg/day, n=6/group) orally in drinking water for 6 weeks. Lesion macrophage content was measured by immunohistochemical staining using monoclonal anti-mouse MOMA2 antibody as a specific marker for monocyte/macrophage in aortic root sections. HAEC LDL oxidation, VCAM-1 expression, and adhesion assays were done by measuring thiobarbituric acid reactive substances (TBARS), Real-time PCR analysis, ELISA, and adhesion rate using fluorescently labeled monocytes respectively. Results and Conclusions: Rev-D4F significantly (p=0.0009) reduced macrophage content in lesions by 47% compared to controls. L-4F mimetic peptide had no effect on macrophage content. In-vitro studies indicated that Rev D-4F significantly inhibited endothelial cell LDL oxidation, TNF-α- and LPS-induced VCAM-1, and oxidized LDL-induced monocyte adhesion. The data suggest that Rev-D4F, through inhibiting inflammatory and LDL oxidative events, reduces endothelial monocyte migration, lesion macrophage content and atherosclerosis.
In the past few decades, the pre‐stressed anchor system has been extensively used to increase the... more In the past few decades, the pre‐stressed anchor system has been extensively used to increase the stability of slope among the Taiwan Island. Several disasters have occurred that caused by the sudden failure of pre‐stressed anchor system. This article presents a real‐time ...
Journal of cardiovascular pharmacology and therapeutics, 2012
Synthetic class A amphipathic helical peptide analogs of apolipoprotein-AI (apoAI; with varied ph... more Synthetic class A amphipathic helical peptide analogs of apolipoprotein-AI (apoAI; with varied phenylalanine residues) are emerging therapeutic approaches under investigation for atherosclerosis. Utilizing retroinverso sequencing, we designed reverse-D4F (Rev-D4F) peptide with 18 d-amino acids containing 4 phenylalanine residues and reverse order that allows the side chain residues to be of exact alignment and superimposable to those of the parent l-amino acid peptide. This study examined the effect of Rev-D4F on atherosclerosis in apolipoprotein E (apoE)-null mice and the underlying mechanisms. ApoE-null mice were fed a chow diet and administered water (control), Rev-D4F, or L4F mimetic peptides (0.4 mg/mL, equivalent to 1.6 mg/d) orally in drinking water for 6 weeks. Aortic root atherosclerotic lesion area, lesion macrophage content, and the ability of plasma high-density lipoprotein (HDL) to influence monocyte chemotaxis were measured. Rev-D4F significantly decreased aortic sinus...
The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal ce... more The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20‐ and 1.3‐fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and i...
Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably e... more Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity.
Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It ha... more Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It has antihyperglycemic activity from inhibition of DPPIV but also striking anticancer activity and a toxicity for which the mechanisms are unknown. 1 cyclizes at physiological pH, which attenuates its inhibitory potency >100-fold, which is a "soft drug" effect. Here we show that this phenomenon can be exploited to create prodrugs with unique properties and potential for selective in vivo targeting. Enzyme-mediated release delivers 1 to the target in the active form at physiological pH; cyclization attenuates systemic pharmacological effects from subsequent diffusion. This "pro-soft" design is demonstrated with a construct activated by and targeted to DPPIV, including in vivo results showing improved antihyperglycemic activity and reduced toxicity relative to 1. Pro-soft derivatives of 1 can help to illuminate the mechanisms underlying the three biological activities, or to help localize 1 at a tumor and thereby lead to improved anticancer agents with reduced toxicity. The design concept can also be applied to a variety of other boronic acid inhibitors.
Efficient syntheses of 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine and 4‐carboxy‐l‐phenylalani... more Efficient syntheses of 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine and 4‐carboxy‐l‐phenylalanine within the context of the pentapeptide Ac‐Ile‐X‐Gly‐Glu‐Phe‐NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide‐based tyrosine kinase inhibitors. The key intermediate, Ac‐Ile‐Phe(4‐formyl)‐Gly‐Glu(O‐tBu)‐Phe‐NH2, was synthesized by a facile palladium‐catalyzed carbonylation of Ac‐Ile‐Phe(4‐iodo)‐Gly‐Glu(O‐tBu)‐Phe‐NH2. Oxidation of Ac‐Ile‐Phe(4‐formyl)‐Gly‐Glu(O‐tBu)‐Phe‐NH2 with tetrabutylammonium permanganate or addition of di‐t‐butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4‐carboxy‐l‐phenylalanine or 4‐(R,S‐hydroxyphosphonomethyl)‐l‐phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c‐src than the corresponding pentapeptide wherei...
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