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1.  Synthesis of Lithocholic Acid Derivatives as Proteasome Regulators 
ACS Medicinal Chemistry Letters  2012;3(11):925-930.
Accumulation of aberrant protein aggregates, such as amyloid β peptide (Aβ), due to decreased proteasome activities, might contribute to the neurodegeneration in Alzheimer's disease. In this study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of the proteasome at an EC50 of 7.8 and 4.3 μM, respectively. Replacing the C24 methyl ester in 2 with methylamide resulted in a complete devoid of proteasome activating activity. Epimerizing the C3 substituent from an α to β orientation transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2 was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the inhibitory effect of Aβ on the proteasome.
PMCID: PMC3544189  PMID: 23330053
proteasome activator; lithocholic acid; Alzheimer's disease; amyloid β
2.  Synthesis of Lithocholic Acid Derivatives as Proteasome Regulators 
ACS medicinal chemistry letters  2012;3(11):925-930.
Accumulation of aberrant protein aggregates, such as amyloid beta peptide (Aβ), due to decreased proteasome activities might contribute to the neurodegeneration in Alzheimer's disease. In this study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of the proteasome at an EC50 of 7.8 and 4.3 μM, respectively. Replacing the C24 methyl ester in 2 with methylamide resulted in a complete devoid of proteasome activating activity. Epimerizing the C3 substituent from an alpha to beta orientation transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2 was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the inhibitory effect of Aβ on the proteasome.
PMCID: PMC3544189  PMID: 23330053
proteasome activator; lithocholic acid; Alzheimer's disease; amyloid beta
3.  Lithocholate glucuronide is a cholestatic agent. 
Journal of Clinical Investigation  1984;73(6):1507-1514.
Lithocholic acid and its taurine, glycine, and sulfate derivatives are potent cholestatic agents. Lithocholate glucuronide is present in the plasma and urine of patients with cholestatic syndromes, but little is known of its metabolism, excretion, and cholestatic potential. [3 beta-3H]lithocholate 3-O-beta-D-glucuronide was synthesized, and chemical and radiochemical purity were established. The aqueous solubility of lithocholate glucuronide was determined and found to be greater than that of lithocholic acid or several of its derivatives. In the range of concentrations examined, calcium ions precipitated lithocholate glucuronide stoichiometrically. The material was administered to rats prepared with an external biliary fistula. When 17-25 micrograms quantities were administered, 89.1 +/- 4.5% (mean +/- SEM) of the radiolabel was secreted in bile within the first 20 h after administration, the major fraction being secreted in less than 20 min. Four-fifths of the radiolabeled material in bile was the administered unaltered parent compound, while a minor fraction consisted of a more polar derivative(s). We showed that increasing biliary concentrations of more polar derivatives were observed with milligram doses of [3H]lithocholate glucuronide, and with time after the administration of these loading doses. Milligram doses of [3H]lithocholate glucuronide resulted in partial or complete cholestasis. When induced cholestasis was partial, secretion in bile remained the primary excretory route (82.5-105.6% recovery in bile), while, when complete cholestasis was induced, wide tissue distribution of radiolabel was observed. Cholestasis developed rapidly during infusion of [3H]lithocholate glucuronide. Bile flow was diminished within 10-20 min of the start of an infusion of 0.05 mumol, 100 g-1 body weight, minute-1, administered concomitantly with an equimolar infusion of taurocholate. The results establish that lithocholate glucuronide exerts cholestatic effects comparable to those exerted by unconjugated lithocholic acid.
PMCID: PMC437060  PMID: 6547150
4.  Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor 
ChemMedChem  2012;7(6):1071-1083.
The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions.
PMCID: PMC3677030  PMID: 22529030
Protein-protein interactions; Structure-activity relationships; Surface plasmon resonance; Steroids; Drug design
Human UDP-glucuronosyltransferase (UGT) 2B7 is the major isoform catalyzing the glucuronidation of a variety of endogenous compounds including bile acids. To determine the role of bile acids in the regulation of UGT2B7 expression, Caco-2 cells were incubated with the natural human farnesoid X receptor (hFXR) ligand, chenodeoxycholic acid, as well as the secondary bile acid, lithocholic acid, derived from chenodeoxycholic acid. Incubation of Caco-2 cells with lithocholic acid in the absence of exogenous hFXR resulted in a dose-dependent down-regulation of UGT2B7 mRNA levels, with an IC50 of 13 μM. Similar down-regulation was also observed with chenodeoxycholic acid; however, much higher concentrations were required. Transient transfection of Caco-2 cells with hFXR suppressed UGT2B7 mRNA expression both in the absence and presence of ligand. UGT2B7 promoter transfection experiments and deletion/mutation analysis showed that lithocholic acid-activated hFXR decreased UGT2B7 promoter activity via a negative hFXR response element (NFRE) located between nucleotides −148 and −134. Cotransfection with hFXR and/or human retinoid X receptor further enhanced the repression. Electrophoretic mobility shift assays additionally confirmed the role of NFRE in UGT2B7 down-regulation by lithocholic acid. These findings suggest that lithocholic acid, an activator of nuclear hFXR, acts as a negative regulator of UGT2B7 expression, indicating that hFXR may play an essential role in lithocholic acid homeostasis through negative regulation of this UGT that is involved in lithocholic acid biotransformation. Therefore, it is postulated that lithocholic acid toxicity may be due to down-regulation of genes involved in its detoxification, including UGT2B7, leading to limited excretion of lithocholic acid from the body.
PMCID: PMC2652669  PMID: 15821044
6.  Amino acid conjugates of lithocholic acid as antagonists of the EphA2 receptor 
Journal of medicinal chemistry  2013;56(7):2936-2947.
The Eph receptor–ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The L-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small molecule antagonists of the EphA2 receptor.
PMCID: PMC3953198  PMID: 23489211
7.  Potentiation of Temozolomide Cytotoxicity by Polymerase β Inhibition Is Increased in the Absence of BRCA2 
Cancer research  2009;70(1):409-417.
Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly utilized in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid, an inhibitor of the key BER enzyme, DNA polymerase β, in cells deficient in expression of the homology-dependent repair factor, BRCA2. In vitro studies show that lithocholic acid suppresses the DNA polymerase and 5′dRP lyase activities of DNA polymerase β by preventing the formation of a stable pol β-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that lithocholic acid exhibits synergism with the alkylating agent, temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both lithocholic acid and temozolomide individually. The potentiation of temozolomide cytotoxicity by lithocholic acid owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by γH2AX immunofluorescence. Death appears to be induced in co-treated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.
PMCID: PMC2943728  PMID: 20028873
8.  Adsorption of Lithocholic Acid to Fusarium equiseti M41 as an Essential Process in Its Conversion to Ursodeoxycholic Acid 
Fusarium equiseti M41 converts lithocholic acid to ursodeoxycholic acid. Adsorption of lithocholic acid particles to mycelia of F. equiseti M41 is essential in the conversion of lithocholic acid to ursodeoxycholic acid. Production of ursodeoxycholic acid was negligible when particles of lithocholic acid were absent. As the concentration of lithocholic acid particles increased, both the amount of mycelium-bound lithocholic acid and the production of ursodeoxycholic acid increased hyperbolically (K1/2 = 1.9 g/liter and Kmapparent = 1.9 g/liter. A fluorescent lithocholic acid derivative was used to confirm that insoluble particles of lithocholic acid attached to the surface of the mycelia. The hydrophobic nature of this binding was estimated from the close relationship observed between the hydrophobicity of bile acids and their binding capacity to the mycelia. By repeated washing with 30% dimethyl sulfoxide, two binding modes of lithocholic acid were distinguished, i.e., surface binding (59% of bound lithocholic acid) and tight binding (41% of bound lithocholic acid). From the amount of tightly bound lithocholic acid, the intracellular concentration of lithocholic acid was calculated to be 1,433-fold higher than its saturating concentration in the reaction mixture, thus promoting effective conversion to ursodeoxycholic acid in the mycelia. Several lines of evidence indicated that glycoproteins of the cell wall participated in the binding of lithocholic acid.
PMCID: PMC202523  PMID: 16347578
In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used.
PMCID: PMC4197193  PMID: 22445925
Fatty acid; Triacylglycerol and Cholesterol Synthesis; Proteasome inhibitor
10.  Discovery and Synthesis of Hydronaphthoquinones as Novel Proteasome Inhibitors 
Journal of medicinal chemistry  2012;55(5):1978-1998.
Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed and compound 1 was synthesized in-house to confirm the structure and activity (IC50 = 3.0 ± 1.6 μM [n=25]). Novel hydronaphthoquinone derivatives of the hit 1 were designed, synthesized and evaluated as proteasome inhibitors. The structure activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties were the most active compounds with up to 20-fold greater CT-L inhibitory than compound 1 (compounds 15e, 15f, 15h 15j, IC50 values around 200 nM and compound 29, IC50 = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC50 = 0.44 to 1.01 μM) than 1 (IC50 = 3.54 to 7.22 μM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analog 39 inhibited proteasome CT-L activity irreversibly.
PMCID: PMC3530929  PMID: 22220566
11.  Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II 
The Journal of Cell Biology  1994;127(2):401-410.
We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.
PMCID: PMC2120198  PMID: 7929584
12.  Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome 
Science (New York, N.Y.)  2016;351(6275):10.1126/science.aad9421 aad9421.
Structured Abstract
The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1).
We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates.
Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2.
A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity.
A ligand-binding hotspot in the proteasome for assembling substrates and cofactors
Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure.
Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
PMCID: PMC4980823  PMID: 26912900
13.  Interaction of Native Bile Acids with Human Apical Sodium Dependent Bile Acid Transporter (hASBT): Influence of Steroidal Hydroxylation Pattern and C-24 Conjugation 
Pharmaceutical research  2006;23(7):1451-1459.
The human Apical Sodium-dependent Bile Acid Transporter (hASBT) is a potential target for drug delivery, but an understanding of hASBT substrate requirements is lacking. The objective of this study was to characterize hASBT interaction with its native substrates, bile acids, including an evaluation of C-24 conjugation and steroidal hydroxylation on transport affinity and inhibition potency.
Transport and inhibition kinetics of 15 bile acids were evaluated (cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate, and lithocholate, including their glycine and taurine conjugates) using an hASBT-Madin-Darby canine kidney (MDCK) monolayer assay. Samples were analyzed via LC-MS or LC-MS-MS.
C-24 conjugation improved the inhibitory potency of all native bile acids. There was an inverse association between number of steroidal hydroxyl groups and inhibitory potency. Glycolithocholate and taurolithocholate were the most potent inhibitors. Results from transport studies followed trends from inhibition studies. Conjugated dihydroxy and monohydroxy bile acids exhibited the highest hASBT-mediated transport (i.e. lower Kt and higher Jmax). Across the 15 bile acids, Kt generally followed Ki. Additionally, Jmax correlated with Ki, where greater inhibition potency was associated with higher transport capacity.
C-24 conjugation and steroidal hydroxylation pattern modulated native bile acid interaction with hASBT, with C-24 effect dominating steroidal hydroxylation effect. Results indicate that bile acid binding to hASBT may be the rate limiting step in the apical transport of bile acids.
PMCID: PMC2882938  PMID: 16783481
14.  Synthesis, Structure-Activity Relationship, and Mechanistic Investigation of Lithocholic Acid Amphiphiles for Colon Cancer Therapy 
MedChemComm  2014;6(1):192-201.
We report a structure-activity relationship of lithocholic acid amphiphiles for their anticancer activities against colon cancer. We synthesized ten cationic amphiphiles differing in nature of cationic charged head groups using lithocholic acid. We observed that anticancer activities of these amphiphiles against colon cancer cell lines are contingent on nature of charged head group. Lithocholic acid based amphiphile possessing piperidine head group (LCA-PIP1) is ~10 times more cytotoxic as compared to its precursor. Biochemical studies revealed that enhanced activity of LCA-PIP1 as compared to lithocholic acid is due to greater activation of apoptosis.LCA-PIP1 induces sub G0 arrest and causes cleavage of caspases. A single dose of lithocholic acid-piperidine derivative is enough to reduce the tumor burden by 75% in tumor xenograft model.
PMCID: PMC4322782  PMID: 25685308
Bile acids; Cancer; Amphiphiles; Anticancer activities; Colon Cancer
15.  The Effects of Boron Derivatives on Lipid Absorption from the Intestine and on Bile Lipids and Bile Acids of Sprague Dawley Rats 
Metal-Based Drugs  1995;2(2):65-72.
N,N-dimethyl-n-octadecylamine borane 1 at 8 mg/kg/day, tetrakis-u-(trimethylamine boranecarboxylato)-bis(trimethyl-carboxyborane)-dicopper(II) 2 at 2.5 mg/kg/day and trimethylamine-carboxyborane 3 at 8 mg/kg/day were evaluated for their effects on bile lipids, bile acids, small intestinal absorption of cholesterol and cholic acid and liver and small intestinal enzyme activities involved in lipid metabolism. The agent administered orally elevated rat bile excretion of lipids, e.g. cholesterol and phospholipids, and compounds 2 and 3 increased the bile flow rate. These agents altered the composition of the bile acids, but there was no significant increase in lithocholic acid which is most lithogenic agent in rats. The three agents did decrease cholesterol absorption from isolated in situ intestinal duodenum loops in the presence of drug. Hepatic and small intestinal mucosa enzyme activities, e.g. ATP-dependent citrate lyase, acyl CoA cholesterol acyl transferase, cholsterol-7-α -hydroxylase, sn glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase, and lipoprotein lipase, were reduced. However, the boron derivatives 1 and 3 decreased hepatic HMG-CoA reductase activity, the regulatory enzyme for cholesterol synthesis, but the compounds had no effects on small intestinal mucosa HMG-CoA reductase activity. There was no evidence of hepatic cell damage afforded by the drugs based on clinical chemistry values which would induce alterations in bile acid concentrations after treatment of the rat.
PMCID: PMC2364959  PMID: 18472747
16.  UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations 
British Journal of Pharmacology  2014;171(23):5195-5208.
Background and Purpose
The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance.
Experimental Approach
UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs.
Key Results
UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects.
Conclusions and Implications
The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.
PMCID: PMC4294034  PMID: 24597515
17.  Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit 
Biochemical Journal  2010;430(Pt 3):461-476.
The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
PMCID: PMC2933030  PMID: 20632995
chymotrypsin-like; immunoproteasome; 26S proteasome; proteasome inhibitor; β5-subunit; ubiquitin–proteasome system (UPS); Ac, acetyl; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HEK, human embryonic kidney; LC50, half-maximal lethal concentration; MPD, 2-methyl-2,4-pentanediol; NFκB, nuclear factor κB; IκB, inhibitory protein of NFκB; NFκB-Luc, NFκB–luciferase; PA, proteasomal activator; PDL, poly-D-lysine; RNAi, RNA interference; siRNA, small interfering RNA; Suc, succinyl; TEV, tobacco etch virus; TNF-α, tumour necrosis factor-α; 4xUb-Luc, tetra-ubiquitin–luciferase; UPS, ubiquitin–proteasome system; Z, benzyloxycarbonyl
18.  α,β-Unsaturated Carbonyl System of Chalcone-Based Derivatives is Responsible for Broad Inhibition of Proteasomal Activity and Preferential Killing of Human Papilloma Virus (HPV)-Positive Cervical Cancer Cells 
Journal of medicinal chemistry  2010;54(2):449-456.
Proteasome inhibitors have potential for the treatment of cervical cancer. We describe the synthesis and biological characterization of a new series of 1,3-diphenylpropen-1-one (chalcone)-based derivatives lacking the boronic acid moieties of the previously reported chalcone-based proteasome inhibitor 3,5-bis-(4-boronic acid-benzylidene)-1-methyl-piperidin- 4-one and bearing a variety of amino acid substitutions on the amino-group of the 4-piperidone. Our lead compound 2 (RA-1) inhibits proteasomal activity and has improved dose-dependent anti-proliferative and pro-apoptotic properties in cervical cancer cells containing human papillomavirus. Further, it induces synergistic killing of cervical cancer cell lines when tested in combination with an FDA approved proteasome inhibitor. Exploration of the potential mechanism of proteasomal inhibition by our lead compound using in silico docking studies suggests that the carbonyl group of its oxopiperidine moiety is susceptible to nucleophilic attack by the γ-hydroxy threonine side chain within the catalytic sites of the proteasome.
PMCID: PMC3204583  PMID: 21186794
Cervical Cancer; Proteasome Inhibitors; Chalcones; Ubiquitin Proteasome; System (UPS); UPS-stress
19.  Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models 
Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes.
There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice.
Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.
PMCID: PMC3206449  PMID: 21992595
20.  Inhibition of the human proteasome by imidazoline scaffolds 
Journal of medicinal chemistry  2013;56(14):10.1021/jm400235r.
The proteasome has emerged as the primary target for the treatment of multiple myeloma. Unfortunately, nearly all patients develop resistance to competitive-type proteasome inhibitors, such as bortezomib. Herein, we describe the optimization of non-competitive proteasome inhibitors to yield derivatives that exhibit nanomolar potency (compound 46, IC50 130 nM) towards proteasome inhibition and overcome bortezomib resistance. These studies illustrate the feasibility of the development of non-competitive proteasome inhibitors as additives and/or alternatives to competitive proteasome inhibitors.
PMCID: PMC3844044  PMID: 23789888
Imidazolines; proteasome; non-competitive inhibition; multiple myeloma; chemotherapy
21.  Tyrosine Nitration of PA700 Activates the 26S Proteasome to Induce Endothelial Dysfunction in Mice With Angiotensin II–Induced Hypertension 
Hypertension  2009;54(3):625-632.
The ubiquitin-proteasome system has been implicated in oxidative stress–induced endothelial dysfunction in cardiovascular diseases. However, the mechanism by which oxidative stress alters the ubiquitin-proteasome system is poorly defined. The present study was conducted to determine whether oxidative modifications of PA700, a 26S proteasome regulatory subunit, contributes to angiotensin II (Ang II)–induced endothelial dysfunction. Exposure of human umbilical vein endothelial cells to low concentrations of Ang II, but not vehicle, for 6 hours significantly decreased the levels of tetrahydro-L-biopterin (BH4), an essential cofactor of endothelial NO synthase, which was accompanied by a decrease in GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. In addition, Ang II increased both tyrosine nitration of PA700 and the 26S proteasome activity, which were paralleled by increased coimmunoprecipitation of PA700 and the 20S proteasome. Genetic inhibition of NAD(P)H oxidase or administration of uric acid (a peroxynitrite scavenger) or NG-nitro-L-arginine methyl ester (nonselective NO synthase inhibitor) significantly attenuated Ang II–induced PA700 nitration, 26S proteasome activation, and reduction of GTP cyclohydrolase I and BH4. Finally, Ang II infusion in mice decreased the levels of both BH4 and GTP cyclohydrolase I and impaired endothelial-dependent relaxation in isolated aortas, and all of these effects were prevented by the administration of MG132, a potent inhibitor for 26S proteasome. We conclude that Ang II increases tyrosine nitration of PA700 resulting in accelerated GTP cyclohydrolase I degradation, BH4 deficiency, and consequent endothelial dysfunction in hypertension.
PMCID: PMC2910588  PMID: 19597039
angiotensin II; proteasome; hypertension; oxidative stress; endothelial NO synthase; tetrahydrobiopterin; GTP cyclohydrolase I
22.  Halophilic 20S Proteasomes of the Archaeon Haloferax volcanii: Purification, Characterization, and Gene Sequence Analysis 
Journal of Bacteriology  1999;181(18):5814-5824.
A 20S proteasome, composed of α1 and β subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa α1β-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75°C. The α1β-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the α1β-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active α1β-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the β-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (Ki = 0.2 and 8 μM, respectively). In addition to the genes encoding the α1 and β subunits, a gene encoding a second α-type proteasome protein (α2) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the α1β-proteasome resulted in the copurification of the α2 protein with the α1 and β subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the α1β-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different α subunits in this halophilic archaeon.
PMCID: PMC94104  PMID: 10482525
23.  Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells 
PLoS ONE  2009;4(4):e5229.
Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death.
Methodology/Principal Findings
HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded α-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (β1 and β5) and structural subunits (α2, α3, α6 and β3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells.
The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.
PMCID: PMC2664966  PMID: 19365565
24.  Synthetic analogs of green tea polyphenols as proteasome inhibitors. 
Molecular Medicine  2002;8(7):382-392.
BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.
PMCID: PMC2040000  PMID: 12393936
25.  Lithocholic acid induction of the FGF19 promoter in intestinal cells is mediated by PXR 
AIM: To study the effect of the toxic secondary bile acid lithocholic acid (LCA) on the expression of fibroblast growth factor 19 (FGF19) in intestinal cells and to characterize the pregnane-X-receptor (PXR) response of the FGF19 promoter region.
METHODS: The intestinal cell line LS174T was stimulated with various concentrations of chenodeoxy-cholic acid and lithocholic acid for several time points. FGF19 mRNA levels were determined with quantitative realtime RT-PCR. FGF19 deletion promoter constructs were generated and the LCA response was analzyed in reporter assays. Co-transfections with PXR and RXR were carried out to study FGF19 regulation by these factors.
RESULTS: LCA and CDCA strongly up-regulate FGF19 mRNA expression in LS174T cells in a time and dose dependent manner. Using reporter gene assays with several deletion constructs we found that the LCA responsive element in the human FGF19 promoter maps to the proximal regulatory region containing two potential binding sites for PXR. Overexpression of PXR and its dimerization partner retinoid X receptor (RXR) and stimulation with LCA or the potent PXR ligand rifampicin leads to a significant induction of FGF19 promoter activity in intestinal cells.
CONCLUSION: LCA induced feedback inhibition of bile acid synthesis in the liver is likely to be regulated by PXR inducing intestinal FGF19 expression.
PMCID: PMC4250623  PMID: 17696253
Pregnane X receptor; Detoxification; Fibroblast growth factor 19; Lithocholic acid

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