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, secondgeneration 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. Cancer Res; 70(5); 1970-80. ©2010 AACR.

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 ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

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.

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2).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.Post-translational modification by ubiquitin plays an essential role in a wide range of cellular processes. One of the most intensively studied pathways is the ubiquitin-proteasome system that attaches a polyubiquitin chain to a lysine residue on a target protein and directs it to proteasome-mediated proteolysis. The ubiquitin-proteasome system is a key system responsible for maintaining cellular protein homeostasis, an emerging research area that could potentially transform our understanding of human diseases (1-3). Bortezomib (VELCADE), a proteasome inhibitor, is currently approved in the treatment of patients with multiple myeloma and relapsed mantle cell lymphoma (4, 5). The clinical success of bortezomib suggests that targeting other components in the ubiquitin-proteasome system pathway might represent an opportunity to develop novel anti-cancer therapeutics (6 -9).Conjugating ubiquitin to a protein substrate is mediated by an enzymatic cascade initiated by ubiquitin-activating enzyme (UAE) 2 (or Ube1 in humans, also known as E1) (10). Previous mechanistic studies show that, in vitro, UAE activates ubiquitin by a three-step process using ATP as a cofactor (Fig. 1A) (11,12). In step 1, UAE binds ATP and ubiquitin, catalyzes formation of ubiquitin adenylate intermediate, and releases inorganic pyrophosphate (PP i ). The ubiquitin adenylate activates the C-terminal carboxyl group of ubiquitin for nucleophilic substitution. In step 2, the catalytic cysteine residue in UAE attacks the adenylate to form a thioester intermediate (UAE-Sϳubiq-uitin, ϳ denotes the thioester bond between the C-terminal carboxyl group of ubiquitin and Cys 632 of human UAE) with AMP as the by-product. In step 3, UAE-Sϳubiquitin binds another equivalent of ATP and ubiquitin and in a second round of adenylation, forms a UAE-Sϳubiquitin⅐ubiquitin-adenylate ternary complex. Although UAE-Sϳubiquitin is capable of transferring ubiquitin to the conjugating enzyme (E2) via a transthiolation reaction, E1-E2 transthiolation is grea...

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 β5 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.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capac change) of ligand binding to FK506 binding protein First suggested >70 years ago (1, 2) and particularly emphasized by Pauling et al. (3,4), the hydrogen bond is now recognized as an interaction of fundamental importance in determining the structures of proteins and their complexes with ligands (5). Nevertheless, conflicting views are still held on the relative energetic contributions ofhydrogen bonds and interactions involving nonpolar groups to the thermodynamics of protein folding and ligand binding (6-8). Models have been presented arguing that the overall enthalpic contribution of interactions involving nonpolar moieties (at 250C) to the process of protein folding is highly unfavorable (7, 9), highly favorable (8), or essentially zero (10, 11). Arguments have also been advanced stating that the overall contribution of amide hydrogen-bond formation to the enthalpy of protein folding (or ligand-protein binding) at 250C is favorable (7), negligible (12) and possibly unfavorable (8,13). Clarification of the present controversy requires estimates of the enthalpies of formation of specific hydrogen bonds in particular regions of known protein structures. Such measurements are essential for understanding the energetic basis of protein folding and for obtaining structure-based predictions of the energies of ligand binding for the purpose of drug design.Part of the difficulty in dissecting the relative energetic contribution of hydrogen bonds to folding or binding reactions of proteins is that the formation of hydrogen bonds between atoms in reaction products is often accompanied by the release ofwater molecules that were hydrogen-bonded to these atoms prior to reaction. Tacrolimus (also known as FK506) and rapamycin are large macrocyclic compounds each of which binds with high affinity to a common cytosolic protein of 12 kDa known as FK506 binding protein . Recently, the structures of FKBP-12 in solution and in the crystalline state have been determined (14, 15), as have the crystal structures of the tacrolimus-FKBP-12 and rapamyci-FKBP-12 complexes (16-18). One of the structural features common to both protein-ligand complexes is a hydrogen bond between the Tyr-82 hydroxyl hydrogen (donor) and the amide carbonyl oxygen (acceptor) of either rapamycin or tacrolimus, buried in the hydrophobic interface (Fig. 1). As determined by x-ray crystallography, an important feature of the unliganded protein structure is the welldefined hydration of this tyrosine hydroxyl group. Furthermore, the NMR structure of tacrolimus in chloroform has been reported by Karuso et al. (19); the low solubility of tacrolimus prevents the determination of its structure in water. However, the interaction of unbound tacrolimus with water has been investigated through molecular dynamics simulation (20). The simulation reveals that the amide carbonyl oxygen of tacrolimus is exposed to solvent and makes an average of 1.6 hydrogen bonds with water mol...

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]