Supplementary Fig. S1 from Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib

Supplementary Fig. S2 from Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.

Inhibition of the proteasome by covalent inhibitors is a clinically proven anti-cancer therapy. We report here that dipeptides with a P3 neopentyl Asn residue are potent, reversible, non-covalent inhibitors selective for the chymotryptic activity of the 20S proteasome in vitro and in cells. The X-ray structure of compound 20 in complex with yeast 20S reveals the importance of hydrophobic bonding interactions of the neopentyl group within the S3 binding pocket of the 20S beta 5 sub-unit. Four compounds show comparable potencies to boronic acid inhibitors in a panel of assays.

The NEDD8 conjugation pathway is initiated by the NEDD8 El, also known as NEDD8 activating enzyme (NAB) or APPBP1/UBA3 (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). The best described biological role for NEDD8 conjugation is to regulate the activity of the cullin RING ligase (CRL) family of ubiquitin E3 ligases (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). In this way, the NEDD8 pathway regulates the turnover of a subset of ubiquitin proteasome system (UPS) substrates that are essential for cancer cell growth and survival (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). We recently initiated clinical trials with a first-in-class small molecule inhibitor of NAB for the treatment of cancer (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). Here we describe a biochemical and cell-based assay used to identify NAB inhibitors and monitor inhibition of the NEDD8 conjugation pathway.

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.

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

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.

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 β-5/6 active site of y20S. Derivative 25, (β5 IC50=7.4nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.

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.

Abstract 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. [Mol Cancer Ther 2006;5(12):3052–61]

The thermodynamic properties of binding of the substrate, inosine monophosphate (IMP), and the uncompetitive inhibitor, mycophenolic acid, to inosine monophosphate dehydrogenase (IMPDH) were measured. Specifically, the free energy, enthalpy, entropy, and heat capacity changes were determined for each ligation state of the tetrameric enzyme, over a temperature range from 2.5 to 37 degrees C by high-precision titration microcalorimetry. It was discovered that IMP binds to IMPDH in a negatively cooperative fashion and that mycophenolic acid binding is critically dependent on the presence of IMP. Moreover, the binding of IMP is entropically driven at low temperatures and enthalpically driven at high temperatures, with an unusually large, temperature dependent heat capacity change. A thermodynamic argument, based on the general nature of the heat capacity function for a binding reaction and its temperature dependence, is used to infer the existence of an equilibrium mixture of at least two structural forms of apo-IMPDH. The equilibrium is perturbed in the presence of IMP and mycophenolic acid, suggesting a mechanism for the ligand-linked conformational changes. An allosteric model, incorporating subunit-subunit interactions nested within a concerted conformational change involving the entire tetrameric macromolecule, is proposed to account for the observed binding behavior. The implications of these findings for the design of novel "allosteric-effector" inhibitors of IMPDH, to be used for the purpose of immunosuppression, are discussed.

The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atoms within the proteins and ligands and of these atoms with water. Delineation of the various energetic contributions of the stabilities of proteins or protein-ligand complexes in aqueous solution, and an evaluation of their structural basis, requires a direct account of the changes, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamycin, each bind with high affinity to a common site on a small FK506 binding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isomerase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the enthalpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solutions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titration calorimetry in the temperature range 5-30-degrees-C. For both tacrolimus and rapamycin binding, a large enthalpic destabilization of binding is observed in D2O relative to H2O, in the temperature range examined. Additionally, large negative constant pressure heat capacity changes are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determinants of the differences in the energetics of binding in light and heavy water. The analysis suggests that a chief contributor to the observed enthalpic destabilization is the differential hydration, of protein and ligand atoms, by light and heavy water.