Darshan S. Sappal

Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-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 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.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.

Genome-Wide siRNA Screen for Modulators of Cell Death Induced by Proteasome Inhibitor Bortezomib

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.

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 β-5/6 active site of y20S. Derivative 25, (β5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.

The antiproliferative agent MLN944 preferentially inhibits transcription.

MLN944 is a novel compound currently being codeveloped by Millennium Pharmaceuticals and Xenova Ltd. as a cancer therapeutic and is in a phase I clinical trial for solid tumors. Although MLN944 was originally proposed to function as a topoisomerase I and II inhibitor, more recent data has shown that it is a DNA-intercalating agent that does not inhibit the catalytic activity of topoisomerase I or II. We show here that MLN944 inhibits incorporation of radiolabeled precursors into RNA preferentially over incorporation into DNA and protein in HCT116 and H460 cells. To determine if MLN944 inhibits transcription, a human RNA polymerase II in vitro transcription system was used. MLN944 inhibited initiation when added before or after the formation of preinitiation complexes and inhibited elongation at higher concentrations. The preferential inhibition of initiation differentiates MLN944 from actinomycin D, which more strongly inhibits elongation. Transcription of all RNA polymerases was inhibited in nuclei isolated from HeLa cells treated with low concentrations of MLN944. Our data are consistent with transcription as the target of the potent cytotoxic effects of MLN944.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

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.

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

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 β5 sub-unit. Four compounds show comparable potencies to boronic acid inhibitors in a panel of assays.

Abstract A164: The small molecule UAE inhibitor TAK-243 (MLN7243) prevents DNA damage repair and reduces cell viability/tumor growth when combined with radiation, carboplatin and docetaxel

Clinical results of VELCADE® (bortezomib) For Injection have prompted evaluation of other enzymes within the ubiquitin proteasome system (UPS) as druggable targets for human cancer. We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), an essential cellular enzyme responsible for activating > 99% of all cellular ubiquitin. Ubiquitin is involved in multiple cellular processes including ubiquitin-dependent protein turnover, cell cycle progression, regulation of apoptosis, protein localization and response to DNA damage. Experiments combining targeted siRNA knockdown with TAK-243 identified DNA damage repair genes necessary for UAE inhibitor-induced cell death. A more focused approach revealed TAK-243 treatment blocked essential monoubiquitination events within the Translesion synthesis (TLS), Fanconi Anemia (FA) and Homologous recombination (HR) pathways. Inhibition of UAE prevented mono-ubiquitin signaling of key mediators within these pathways, including PCNA and FANCD2, by blocking formation of their specific E2-ubiquitin thioesters. In vitro cell-based assays combining TAK-243 with ultraviolet (UV) and radiation, both known to induce DNA damage, yielded inhibition of cell growth and enhanced DNA damage as observed through colony formation assays and Comet assay detection, respectively. Xenograft tumor bearing mice were treated with carboplatin or docetaxel, combined with TAK-243, to evaluate combination benefits in vivo. Synergistic and additive anti-tumor combination benefits were observed in animals treated with TAK-243 + carboplatin and TAK-243 + docetaxel. These important mechanistic in vitro and in vivo studies indicate the dependency of ubiquitination signaling in DNA damage repair and provide a mechanistic rationale for combining radiation, carboplatin or docetaxel with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (ClinicalTrials.gov identifier: NCT02045095). Citation Format: Michael A. Milhollen, Judi Shi, Tary Traore, Jessica Huck, Darshan Sappal, Jennifer Duffy, Eric Lightcap, Yuko Ishii, Jeff Ciavarri, Paul Fleming, Neil Bence, Marc L. Hyer. The small molecule UAE inhibitor TAK-243 (MLN7243) prevents DNA damage repair and reduces cell viability/tumor growth when combined with radiation, carboplatin and docetaxel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A164.

Abstract C82: Identification and preclinical characterization of inhibitors of the ubiquitin-activating enzymes UBA1 and UBA6.

Millennium Pharmaceuticals, Inc. is dedicated to the discovery and development of novel oncology therapeutics in the area of protein homeostasis. Here we report the identification and characterization of compounds that target the ubiquitin activating enzymes, UBA1 and UBA6. These compounds are mechanism based inhibitors that inactivate the ubiquitin E1 enzymes by forming a ubiquitin compound adduct that remains tightly associated with the E1 adenylate binding site. Treatment of cells with these inhibitors results in cellular effects consistent with known Uba1 biology including rapid loss of E2 ubiquitin thioesters, loss of total ubiquitin conjugates, and accumulation of many ubiquitin proteasome system substrates. Following prolonged treatment, cells primarily arrest in the G2 phase of the cell cycle and ultimately undergo apoptosis. Reflecting the extensive cellular roles of ubiquitin, the compounds also impact global protein turnover, ER stress and DNA damage repair. UBA1 inhibition impairs ubiquitination of PCNA and the Fanconia Anemia protein FANCD2 leading to defective repair of UV induced DNA damage. UBA1 inhibition impacts numerous biological pathways relevant to cancer, results in apoptosis in vitro and is capable of inhibiting tumor growth in mouse xenografts in vivo. These data implicate UBA1 as a target for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C82.

Abstract A70: A genome‐wide siRNA screen for modulators of cell death induced by the proteasome inhibitor bortezomib

Multiple pathways have been proposed as the mechanism by which proteasome inhibition induces cell death. To clarify their relative importance, we performed a genome‐wide siRNA screen to evaluate the genetic determinants that confer sensitivity of the HCT‐116 colon cancer cell line to bortezomib (VELCADE®, PS‐341). The screen identified 100 genes whose knock‐down affects the lethality of bortezomib. From this list, the accumulation of the proteins ASF1B, Myc, ODC1, PMAIP1 (Noxa), BNIP3, Gadd45α, p‐SMC1A, SREBF1, and p53 by proteasome inhibition was linked to the induction of cell death. Fifty‐nine genes in the A375 melanoma cell line and 56 genes in the HeLa cervical cancer cell line showed similar interactions with bortezomib to those seen in HCT‐116 and a subset of 39 genes were common to all three cell lines. Finally, knockdown of these 100 genes in HCT‐116 cells similarly affected their responsiveness to a structurally diverse set of proteasome inhibitors. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A70.