Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (ß1i), MECL-1 (ß2i), and LMP7 (ß5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity.

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.

Identification of potent and selective non-covalent inhibitors of the Plasmodium falciparum proteasome.

We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.

Histone deacetylase inhibitors derived from 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine and related heterocycles selective for the HDAC6 isoform.

Acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide are potent sub-type selective HDAC6 inhibitors. Constrained heterocyclic analogs based on 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine show further enhanced HDAC6 selectivity and inhibitory activity in cells. Homology models suggest that the heterocyclic spacer can more effectively access the wider catalytic channel of HDAC6 compared to other HDAC sub-types.

Why the structure but not the activity of the immunoproteasome subunit low molecular mass polypeptide 2 rescues antigen presentation.

The proteasome is responsible for the generation of most epitopes presented on MHC class I molecules. Treatment of cells with IFN-γ leads to the replacement of the constitutive catalytic subunits ß1, ß2, and ß5 by the inducible subunits low molecular mass polypeptide (LMP) 2 (ß1i), multicatalytic endopeptidase complex-like-1 (ß2i), and LMP7 (ß5i), respectively. The incorporation of these subunits is required for the production of numerous MHC class I-restricted T cell epitopes. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes, but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY(246-254) was dependent on LMP2. Treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY(246-254) presentation, whereas silencing of ß1 activity increased presentation of UTY(246-254). In vitro degradation experiments showed that the caspase-like activity of ß1 was responsible for the destruction of this CTL epitope, whereas it was preserved when LMP2 replaced ß1. Moreover, inhibition of the ß5 subunit rescued the presentation of the influenza matrix 58-66 epitope, thus suggesting that a similar mechanism can apply to the exchange of ß5 by LMP7. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL epitope.

N,C-Capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes: role of S3 and S1 binding pockets.

We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in the S3 and S1 binding pockets appeared to account for the species selectivity. The inhibitors can penetrate mycobacteria and kill nonreplicating M. tuberculosis under nitrosative stress.

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues.

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.

Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S beta5-subunit.

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 based [corrected] 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 beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (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 beta5 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 NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 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 beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

Optimization of a series of dipeptides with a P3 threonine residue as non-covalent inhibitors of the chymotrypsin-like activity of the human 20S proteasome.

Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.

Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: kinetic characterization of NEDD8 activating enzyme.

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous "E1s" initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [32P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [32P]PPi and then measuring [32P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [32P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome.

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.

Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib.

Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated.

Proteasome inhibitors bortezomib and carfilzomib used for the treatment of multiple myeloma do not inhibit the serine protease HtrA2/Omi.

The proteasome inhibitor bortezomib is associated with the development of peripheral neuropathy in patients, but the mechanism by which bortezomib can induce peripheral neuropathy is not fully understood. One study suggested that off-target inhibition of proteases other than the proteasome, particularly HtraA2/Omi, may be the underlying mechanism of the neuropathy. The same study also concluded that carfilzomib, a second proteasome inhibitor that is associated with less peripheral neuropathy in patients than bortezomib, showed no inhibition of HtrA2/Omi. The goal of the work described here was to determine whether either proteasome inhibitors truly affected HtrA2/Omi activity. A variety of methods were used to test the effects of both bortezomib and carfilzomib on HtrA2/Omi activity that included in vitro recombinant enzyme assays, and studies with the human neuroblastoma SH-SY5Y cell line and HtrA2/Omi-knockout mouse embryonic fibroblasts. The compound ucf-101 was used to assess the effects of specific HtrA2/Omi inhibition. In contrast to previously published data, our results clearly demonstrated that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity in recombinant enzyme assays at concentrations up to 100 µM, while the specific inhibitor ucf-101 did inhibit the enzyme. The proteasome inhibitors did not inhibit HtrA2/Omi activity in either SH-SY5Y cells or mouse embryonic fibroblasts, as determined by expression of the HtrA2/Omi substrates eIF4G1 and UCH-L1. Based on our biochemical and cell-based assays, we conclude that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity. Therefore, it is unlikely that bortezomib associated peripheral neuropathy is a direct result of off-target inhibition of HtrA2/Omi.

Preclinical evaluation of the antitumor activity of bortezomib in combination with vitamin C or with epigallocatechin gallate, a component of green tea.

PURPOSE: To investigate whether clinically relevant levels of epigallocatechin gallate (EGCG, a component of green tea) or vitamin C (ascorbic acid) could antagonize bortezomib antitumor activity in CWR22 human prostate xenograft tumors. METHODS: The pharmacokinetics (PK) of EGCG and ascorbic acid were determined in immunocompromised mice and compared with concentrations measured in human PK studies of dietary supplements. Antitumor activity of bortezomib in combination with EGCG or ascorbic acid was determined using several dosing regimens to evaluate different target plasma concentrations of EGCG and ascorbic acid. RESULTS: Bortezomib dosed twice-weekly at 0.8 mg/kg IV demonstrated tumor growth inhibition (TGI) of 53.9-58.9%. However, when combined with EGCG such that the plasma concentrations of EGCG were >200 µM at the time of bortezomib dosing, all antitumor activity was abrogated (TGI = -17.7%). A lower concentration of EGCG (11-16 µM), which is severalfold higher than measured clinically in humans taking EGCG supplements (0.6-3 µM), was not antagonistic to bortezomib (TGI 63.5%). Pharmacodynamic studies of proteasome inhibition reflected these findings. Ascorbic acid (40 and 500 mg/kg PO daily) was evaluated under a similar study design and did not antagonize bortezomib antitumor activity (TGI 57.2 and 72.2%). CONCLUSIONS: No antagonism of bortezomib is seen in preclinical in vivo experiments, where EGCG or ascorbic acid plasma concentrations are commensurate with dietary or supplemental intake. The data suggest that patients receiving bortezomib treatment do not need to avoid normal dietary consumption of green tea, vitamin C-containing foods, or EGCG or vitamin C dietary supplements.

Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer.

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.

Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates.

The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome.

The Caenorhabditis elegans muscle specific serpin, SRP-3, neutralizes chymotrypsin-like serine peptidases.

Members of the intracellular serpin family may help regulate apoptosis, tumor progression, and metastasis. However, their in vivo functions in the context of a whole organism have not been easily defined. To better understand the biology of these serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model organism. Previous in silico analysis suggested that the C. elegans genome harbors nine serpin-like sequences bearing significant similarities to the human clade B intracellular serpins. However, only five genes appear to encode full-length serpins with intact reactive site loops. To determine if this was the case, we have cloned and expressed a putative inhibitory-type C. elegans serpin, srp-3. Analysis of SRP-3 inhibitory activity indicated that SRP-3 was a potent inhibitor of the serine peptidases, chymotrypsin and cathepsin G. Spatial and temporal expression studies using GFP and LacZ promoter fusions indicated that SRP-3 was expressed primarily in the anterior body wall muscles, suggesting that it may play a role in muscle cell homeostasis. Combined with previous studies showing that SRP-2 is an inhibitor of the serine peptidase, granzyme B, and lysosomal cysteine peptidases, these data suggested that C. elegans expressed at least two inhibitory-type serpins with nonoverlapping expression and inhibitory profiles. Moreover, the profiles of these clade L serpins in C. elegans share significant similarities with the profiles of clade B intracellular serpin members in higher vertebrates. This degree of conservation suggests that C. elegans should prove to be a valuable resource in the study of metazoan intracellular serpin function.

Correlation of serpin-protease expression by comparative analysis of real-time PCR profiling data.

Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin-protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin-protease matched expression may underlie the imbalanced protease activity observed in pathological states.

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.