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1.  New Small-Molecule Inhibitor Class Targeting Human Immunodeficiency Virus Type 1 Virion Maturation▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):5080-5087.
A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.
PMCID: PMC2786326  PMID: 19805571
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.  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
4.  New Betulinic Acid Derivatives as Potent Proteasome Inhibitors 
In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 µM, respectively, which are three- to four-fold more potent than the proteasome inhibition controls LLM-F and lactacystin.
PMCID: PMC3171619  PMID: 21856154
5.  The Proteasome Is a Molecular Target of Environmental Toxic Organotins 
Environmental Health Perspectives  2008;117(3):379-386.
Because of the vital importance of the proteasome pathway, chemicals affecting proteasome activity could disrupt essential cellular processes. Although the toxicity of organotins to both invertebrates and vertebrates is well known, the essential cellular target of organotins has not been well identified. We hypothesize that the proteasome is a molecular target of environmental toxic organotins.
Our goal was to test the above hypothesis by investigating whether organotins could inhibit the activity of purified and cellular proteasomes and, if so, the involved molecular mechanisms and downstream events.
We found that some toxic organotins [e.g., triphenyltin (TPT)] can potently and preferentially inhibit the chymotrypsin-like activity of purified 20S proteasomes and human breast cancer cellular 26S proteasomes. Direct binding of tin atoms to cellular proteasomes is responsible for the observed irreversible inhibition. Inhibition of cellular proteasomes by TPT in several human cell lines results in the accumulation of ubiquitinated proteins and natural proteasome target proteins, accompanied by induction of cell death.
The proteasome is one of the molecular targets of environmental toxic organotins in human cells, and proteasome inhibition by organotins contributes to their cellular toxicity.
PMCID: PMC2661907  PMID: 19337512
cell death; molecular target; organotins; proteasome; proteasome inhibitors; TPT
6.  Probing the anticancer mechanism of prospective herbal drug Withaferin A on mammals: a case study on human and bovine proteasomes 
BMC Genomics  2010;11(Suppl 4):S15.
The UPP (ubiquitin proteasome pathway) is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells which regulates cellular events, including mitotis, differentiation, signal transduction, apoptosis, and inflammation. UPP controls activation of the transcriptional factor NF-κB (nuclear factor κB), which is a regulatory protein playing central role in a variety of cellular processes including immune and inflammatory responses, apoptosis, and cellular proliferation. Since the primary interaction of proteasomes occurs with endogenous proteins, the signalling action of transcription factor NF-κB can be blocked by inhibition of proteasomes. A great variety of natural and synthetic chemical compounds classified as peptide aldehydes, peptide boronates, nonpeptide inhibitors, peptide vinyl sulfones and epoxyketones are now widely used as research tools for probing their potential to inhibit proteolytic activities of different proteasomes and to investigate the underlying inhibition mechanisms. The present work reports a bio-computational study carried out with the aim of exploring the proteasome inhibition capability of WA (withaferin A), a steroidal lactone, by understanding the binding mode of WA as a ligand into the mammalian proteasomes (X-ray crystal structure of Bos taurus 20S proteasome and multiple template homology modelled structure of 20S proteasome of Homo sapiens) using molecular docking and molecular dynamics simulation studies.
One possible mode of action which is proposed here for WA to act as a proteasome inhibitor is by suppression of the proteolytic activity which depends on the N-terminal threonine (Thr1) residue hydroxyl group. Docking studies carried out with herbal ligand WA into the structures of bovine and human proteasomes substantiate that WA has the ability to inhibit activity of mammalian 20S proteasomes by blocking the nucleophilic function of N-terminal Thr1. Results from molecular dynamics simulations in water show that the trajectories of both the native human 20S proteasome and the proteasome complexed with WA are stable over a considerably long time period of 4 ns suggesting the dynamic structural stability of human 20S proteasome/WA complex.
Inhibition of proteasomal activity are promising ways to retard or block degradation of specific proteins to correct diverse pathologies. Though quite a number of selective and efficient proteasomal inhibitors exist nowadays, their toxic side effects limit their potential in possible disease treatment. Thus there is an indispensable need for exploration of novel natural products as antitumor drug candidates. The present work supports the mammalian proteasomes inhibiting activity of WA along with elucidation of its possible mode of action. Since WA is a small herbal molecule, it is expected to provide one of the modest modes of inhibition along with added favours of ease in oral administration and decreased immunogenicity. The molecular docking results suggest that WA can inhibit the mammalian proteasomes irreversibly and with a high rate through acylation of the N-terminal Thr1 of the β-5 subunit.
PMCID: PMC3005937  PMID: 21143798
7.  Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells 
Breast Cancer Research  2005;7(6):R897-R908.
A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison.
Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage.
When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis.
The feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper.
PMCID: PMC1410741  PMID: 16280039
8.  Cellular and computational studies of proteasome inhibition and apoptosis induction in human cancer cells by amino acid Schiff base–copper complexes 
Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin–proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base–copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and IκB-α, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi–pi, pi–cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents.
PMCID: PMC3676669  PMID: 23142973
Anticancer; Drug discovery; Molecular modeling; Proteasome inhibitors; Apoptosis; Copper complexes
9.  Inhibition of proteasome activity by the dietary flavonoid apigenin is associated with growth inhibition in cultured breast cancer cells and xenografts 
Proteasome inhibition is an attractive approach to anticancer therapy and may have relevancy in breast cancer treatment. Natural products, such as dietary flavonoids, have been suggested as natural proteasome inhibitors with potential use for cancer prevention and therapeutics. We previously reported that apigenin, a flavonoid widely distributed in many fruits and vegetables, can inhibit proteasome activity and can induce apoptosis in cultured leukemia Jurkat T cells. Whether apigenin has proteasome-inhibitory activity in the highly metastatic human breast MDA-MB-231 cells and xenografts, however, is unknown.
MDA-MB-231 breast cancer cell cultures and xenografts were treated with apigenin, followed by measurement of reduced cellular viability/proliferation, proteasome inhibition, and apoptosis induction. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity, by ubiquitinated proteins, and by accumulation of proteasome target proteins in extracts of the treated cells or tumors. Apoptotic cell death was measured by capase-3/caspase-7 activation, poly(ADP-ribose) polymerase cleavage, and immunohistochemistry for terminal nucleotidyl transferase-mediated nick end labeling positivity.
We report for the first time that apigenin inhibits the proteasomal chymotrypsin-like activity and induces apoptosis not only in cultured MDA-MB-231 cells but also in MDA-MB-231 xenografts. Furthermore, while apigenin has antibreast tumor activity, no apparent toxicity to the tested animals was observed.
We have shown that apigenin is an effective proteasome inhibitor in cultured breast cancer cells and in breast cancer xenografts. Furthermore, apigenin induces apoptotic cell death in human breast cancer cells and exhibits anticancer activities in tumors. The results suggest its potential benefits in breast cancer prevention and treatment.
PMCID: PMC2246179  PMID: 18300387
10.  p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition 
eLife  2014;3:e01856.
Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol.
eLife digest
Cells exposed to high temperatures, infections and other forms of stress often produce oxygen ions and peroxide molecules that can cause damage to proteins and DNA. Cells therefore rely on molecular machines called proteasomes to eliminate damaged proteins, before they cause too much harm. Two related transcription factors—proteins that interact with DNA to ‘switch on’ the expression of genes—are involved in a cell’s responses to stress, but in different ways. Nrf2 switches on genes that limit the damage caused by oxygen ions and peroxide molecules, while Nrf1 switches on the genes that encode the components of the proteasome. As such, Nrf1 helps to restart proteasome activity if it has been shut off—a phenomenon known as ‘bounce-back’.
Within a cell, Nrf1 is known to start off embedded within the membranes of a structure called the endoplasmic reticulum. However, it is not clear how activated Nrf1 leaves this membrane and enters the nucleus to interact with the cell’s DNA. Now, Radhakrishnan et al. show that when Nrf1 is produced, most of its length is found inside the endoplasmic reticulum, with only a small piece being anchored in the surrounding membrane. This is unlike previously described transcription factors that associate with the endoplasmic reticulum, which are stuck to the outside of this structure.
Radhakrishnan et al. also discovered that the activation of Nrf1 depends on an enzyme called p97 or VCP. This enzyme helps to flip Nrf1 from the inside of the endoplasmic reticulum to its outside surface. In most cells, the proteasome then breaks down this part of Nrf1. However, if the proteasome is inhibited, an unknown enzyme cuts Nrf1 free from the endoplasmic reticulum, allowing it to migrate to the nucleus and promote the production of more proteasome components to counteract the inhibition.
Interestingly, drugs that inhibit the proteasome are used to combat cancer because the build-up of damaged proteins is toxic to the cancer cells. By showing that p97 promotes the ‘bounce-back’ of the proteasome, the work of Radhakrishnan et al. suggests that combining existing proteasome inhibitors with drugs that inhibit p97 could eventually lead to new, more effective, therapies for cancer or other diseases.
PMCID: PMC3896944  PMID: 24448410
p97; Nrf1; proteasome; human; mouse
11.  Organic cadmium complexes as proteasome inhibitors and apoptosis inducers in human breast cancer cells 
Although cadmium (Cd) is a widespread environmental contaminant and human carcinogen, our studies indicate an organic Cd complex to be a potent inhibitor of proteasomal chymotrypsin-like (CT-like) activity, further capable of inducing apoptosis in a cancer cell-specific manner. It has been reported that the ligands indole-3-butyric acid (L1) and indole-3-propionic acid (L2) have cancer-fighting effects when tested in a rat carcinoma model. In addition, 3, 5-diaminobenzoic acid o-vanillin Schiff bases (L3) have high antimicrobial activity and a large number of Schiff base complexes have been reported to have proteasome-inhibitory activity. We therefore hypothesized that synthetic forms of Cd in combination with L1, L2 and L3 may have proteasome-inhibitory and apoptosis-inducing activities, which would be cancer cell-specific. To test this hypothesis, we have synthesized three novel Cd-containing complexes: [Cd2(C12H12O2N)4(H2O)2]·2H2O (Cd1), [Cd2(C11H10O2N)4(H2O)2]·2H2O (Cd2) and [Cd(C7H4N2O2)(C8H6O2)2]·2H2O (Cd3), by using these three ligands. We sought out to characterize and assess the proteasome-inhibitory and anti-proliferative properties of these three Cd complexes in human breast cancer cells. Cd1, Cd2 and Cd3 were found to effectively inhibit the chymotrypsin-like activity of purified 20S proteasome with IC50 values of 2.6, 3.0 and 3.3 μM, respectively. Moreover, inhibition of cancer cell proliferation also correlated with this effect. As a result of proteasomal shutdown, the accumulation of ubiquitinated proteins and the proteasome target IκB-α protein as well as induction of apoptosis were observed. To account for the cancer specificity of this effect, immortalized, non-tumorigenic breast MCF10A cells were used under the same experimental conditions. Our results indicate that MCF10A cells are much less sensitive to the Cd1, Cd2 and Cd3 complexes when compared to MDA MB 231 breast cancer cells. Therefore, our study suggests that these Cd organic complexes are capable of inhibiting tumor cellular proteasome activity and consequently induce cancer cell-specific apoptotic death.
PMCID: PMC3648619  PMID: 23499788
Cadmium; Proteasome inhibitor; Apoptosis; Indole-3-butyric acid; Indole-3-propionic acid; 3 5-diaminobenzoic acid o-vanillin Schiff bases
12.  Molecular study on copper-mediated tumor proteasome inhibition and cell death 
The metal ion copper is a cofactor essential for maintaining normal biological and physical functions in human beings. High copper levels have been found in variety of tumor tissues and are involved in tumor angiogenesis processes. The ubiquitin-proteasome system plays an important role in cell growth and apoptosis and has been shown as a novel target for cancer therapy. We previously reported that some organic copper complexes can inhibit the proteasomal chymotrypsin-like activity and induce apoptosis in human cancer cells and xenograft models. In the current study, we investigated the effect of oxidation status of copper, Cu(I) or Cu(II), on inhibition of proteasome activity, induction of apoptosis, and induction of reactive oxygen species (ROS) in human cancer cells. We report four major findings here: i) both Cu(I) and Cu(II) could inhibit the chymotrypsin-like activity of purified 20S proteasome, but Cu(I) was more potent than Cu(II), ii) purified 20S proteasome protein was able to reduce Cu(II) to Cu(I), suggesting that Cu(I) is the oxidation status of copper that directly reacts with the proteasome, iii) when complexed with the copper ligand neocuproine, Cu(I) showed higher ability to induce ROS production in cancer cells, compared with Cu(II), iv) addition of a ROS scavenger in the cancer cell culture-blocked copper-induced ROS generation, but did not overcome copper-mediated proteasome-inhibitory and cell death-inducing events, demonstrating the ROS-independent proteasome-inhibitory property of copper complexes.
PMCID: PMC3809063  PMID: 20514399
copper; oxidation status; proteasome inhibition; apoptosis; ROS
13.  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
14.  Human Immunodeficiency Virus Type 1 Resistance to the Small Molecule Maturation Inhibitor 3-O-(3′,3′-Dimethylsuccinyl)-Betulinic Acid Is Conferred by a Variety of Single Amino Acid Substitutions at the CA-SP1 Cleavage Site in Gag▿ †  
Journal of Virology  2006;80(24):12095-12101.
The compound 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.
PMCID: PMC1676313  PMID: 17035324
15.  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
16.  The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid 
Retrovirology  2004;1:15.
Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells.
Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug.
These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.
PMCID: PMC479704  PMID: 15225375
17.  Inhibitors Selective for Mycobacterial versus Human Proteasomes 
Nature  2009;461(7264):621-626.
Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis (Mtb). Given that proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-ones kill non-replicating Mtb and act as selective suicide-substrate inhibitors of the Mtb proteasome by cyclo-carbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose H-bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-ones to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homolog.
PMCID: PMC3172082  PMID: 19759536
18.  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
19.  Anti-Human Immunodeficiency Virus Activity of YK-FH312 (a Betulinic Acid Derivative), a Novel Compound Blocking Viral Maturation 
Betulinic acid, a triterpenoid isolated from the methyl alcohol extract of the leaves of Syzigium claviflorum, was found to have a potent inhibitory activity against human immunodeficiency virus type 1 (HIV-1). Betulinic acid derivatives were synthesized to enhance the anti-HIV activity. Among the derivatives, 3-O-(3′,3′-dimethylsuccinyl) betulinic acid, designated YK-FH312, showed the highest activity against HIV-induced cytopathic effects in HIV-1-infected MT-4 cells. To determine the step(s) of HIV replication affected by YK-FH312, a syncytium formation inhibition assay in MOLT-4/HIV-1IIIB and MOLT-4 coculture, a multinuclear-activation-of-galactosidase-indicator (MAGI) assay in MAGI-CCR5 cells, electron microscopic observation, and a time-of-addition assay were performed. In the syncytium formation inhibition assay or in the MAGI assay for de novo infection, the compound did not show inhibitory effects against HIV replication. Conversely, no virions were detected in HIV-1-infected cell cultures treated with YK-FH312 either by electron microscopic observation or by viral yield in the supernatant. In accordance with a p24 enzyme-linked immunosorbent assay of culture supernatant in the time-of-addition assay, YK-FH312 inhibited virus expression in the supernatant when it was added 18 h postinfection. However, Western blot analysis of the cells in the time-of-addition assay revealed that the production of viral proteins in the cells was not inhibited completely by YK-FH312. These results suggest that YK-FH312 might affect the step(s) of virion assembly and/or budding of virions, and this is a novel mechanism of action of an anti-HIV compound.
PMCID: PMC90447  PMID: 11257038
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
21.  Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo 
Cancer research  2008;68(18):7283-7292.
Curcumin (diferuloylmethane) is the major active ingredient of turmeric (curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiological conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the N-terminal threonine of the proteasomal chymotrypsin-like subunit. Consistently, curcumin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50=1.85 µM) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor–bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression and apoptosis induction in tumor tissues. Our study demonstrates that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapaeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early stage and late stage/refractory colon cancer.
PMCID: PMC2556983  PMID: 18794115
Curcumin; polyphenols; proteasome inhibitors; colon cancer; apoptosis
22.  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
23.  Proteasome Subtypes and Regulators in the Processing of Antigenic Peptides Presented by Class I Molecules of the Major Histocompatibility Complex 
Biomolecules  2014;4(4):994-1025.
The proteasome is responsible for the breakdown of cellular proteins. Proteins targeted for degradation are allowed inside the proteasome particle, where they are cleaved into small peptides and released in the cytosol to be degraded into amino acids. In vertebrates, some of these peptides escape degradation in the cytosol, are loaded onto class I molecules of the major histocompatibility complex (MHC) and displayed at the cell surface for scrutiny by the immune system. The proteasome therefore plays a key role for the immune system: it provides a continued sampling of intracellular proteins, so that CD8-positive T-lymphocytes can kill cells expressing viral or tumoral proteins. Consequently, the repertoire of peptides displayed by MHC class I molecules at the cell surface depends on proteasome activity, which may vary according to the presence of proteasome subtypes and regulators. Besides standard proteasomes, cells may contain immunoproteasomes, intermediate proteasomes and thymoproteasomes. Cells may also contain regulators of proteasome activity, such as the 19S, PA28 and PA200 regulators. Here, we review the effects of these proteasome subtypes and regulators on the production of antigenic peptides. We also discuss an unexpected function of the proteasome discovered through the study of antigenic peptides: its ability to splice peptides.
PMCID: PMC4279167  PMID: 25412285
antigenic peptides; proteasome; regulator; peptide splicing; processing; CTL
24.  Exploration of the antiplatelet activity profile of betulinic acid on human platelets 
Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including anti-retroviral, antibacterial, antimalarial and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (Adenosine Diphosphate, Thrombin Receptor Activator Peptide-14 and Arachidonic Acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR derived structure of betulinic acid and prostacyclin agonists (PGI2) and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that reserves further investigation.
PMCID: PMC3676635  PMID: 22720759
betulinic acid; platelet aggregation; ADP; antithrombotics
25.  Metal-based 2,3-indolinedione derivatives as proteasome inhibitors and inducers of apoptosis in human cancer cells 
Proliferation and apoptotic pathways are tightly regulated in cells by the ubiquitin-proteasome system (UPS). Alterations in the UPS may result in cellular transformation or other pathological conditions. The proteasome is indeed often found to be overactive in cancer cells. It has been reported that 2,3-indolinedione (L), which exists in marine organisms, as well as in mammals, is a proteasome inhibitor. Studies have shown that metal-based complexes inhibit proteasome activity and induce apoptosis in certain human cancer cells. In the current study, we synthesized six novel metal-based complexes with derivatives of 2,3-indolinedione: [Cd (C15H11O3N2) (CH3COO)] (C1), [Cd (C15H11O2N2) (CH3COO)] (C2), [Co (C15H9O4N2) (CH3COO)] (C3), [Co (C15H11O2N2) (CH3COO)] (C4), [Zn (C19H14O3N3) (CH3COO)] (C5) and [Zn (C17H13O3N2) (CH3COO)] (C6). We sought to characterize and assess the proteasome inhibitory and anti-proliferative effects of these metal-based complexes in human breast (MDA-MB-231) and prostate (LNCaP and PC-3) cancer cells, in order to determine whether specific structures contribute to the inhibition of tumor proteasome activity and the induction of apoptosis. The results revealed that the complexes, C1, C3 and C5, but not their counterparts, C2, C4 and C6, inhibited the chymotrypsin-like activity of the human cancer cellular 26S proteasome; in addition, these complexes promoted the accumulation of the proteasome target protein, Bax, inhibited cell growth and induced apoptosis in a concentration- and time-dependent manner due to their unique structures. Our data suggest that the study of metal-based complexes, including aromatic ring structures with electron-attracting groups, may be an interesting research direction for the development of anticancer drugs.
PMCID: PMC4121346  PMID: 25017797
ubiquitin-proteasome system; metal-based complexes; 2,3-indolinedione derivative; inhibitor; electron-attracting group

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