Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity And Induces Tumor Cell Death

Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity And Induces Tumor Cell Death. ABSTRACT Multiple myeloma (MM) is an aggressive hematopoietic cancer of plasma cells. The recent emergence more than 250-fold higher anti-tumor activities than observed with the natural product syringolin A (SylA). In vivo studies in mice revealed a maximum tolerated dose (MTD) of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.


INTRODUCTION
Multiple Myeloma (MM) is an aggressive hematopoietic cancer of plasma cells that develops in about 6 per 100,000 people per year. MM is considered to be treatable but incurable, with a five year survival rate of 45%. After non-Hodgkin lymphoma, MM is the second most common hematological malignancy in the U.S. and constitutes 1% of all cancers (1). The current therapeutic options include six major drug classes: classic drugs like chemotherapeutic agents, corticosteroids, interferon and immunomodulatory drugs (e.g., thalidomide), and more recently also proteasome inhibitors, histone deacetylase inhibitors (e.g., panobinostat), and the first FDA-approved monoclonal antibody against MM, daratumumab (Darzalex®).
The recent FDA approval of the three proteasome inhibitors bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®) shows that the proteasome is a valuable therapeutic target against neoplastic disease (2,3). Notably, it has been shown that actively dividing cancer cells are more sensitive to proteasome inhibition than quiescent or differentiated normal cells. For example, MM cells are significantly more sensitive to the pro-apoptotic effects of bortezomib (BTZ)-induced proteasome inhibition than are healthy bone marrow cells or peripheral blood mononuclear cells (4). The differential sensitivity of cells to proteasome inhibition may in part be due to the fact that cancer cells require a higher rate of protein turnover than normal cells and, therefore, may be more susceptible to losing proteasome function. Proteasome inhibition also enhances the sensitivity of cancer cells to traditional chemotherapies, providing a rationale for the development of combination therapies.
Despite the fact that proteasome inhibition is a validated strategy for therapy of MM (5-7) and mantle cell lymphoma (8,9), this disease remains challenging as relapses are common and usually associated with increasing chemoresistance (10). Moreover, proteasome inhibitors like BTZ can induce peripheral neuropathy and other toxicities that may decrease efficacy by compromising the ability to deliver therapy at optimal doses. Thus, there are a number of shortcomings and there is an urgent need to develop next generation proteasome inhibitors with improved safety profiles for therapeutic use.
We have discovered and developed a novel class of proteasome inhibitors referred to as syrbactins (11,12). Our previous findings indicated that the natural product syringolin A (SylA) inhibits the proteasome (12)(13)(14)(15) and induces cell death in a number of tumor cell types including MM and neuroblastoma (NB) cells (11,12,14). SylA reacts irreversibly with the N-terminal threonine (Thr) of the active site in the β-5 pocket by a 1,4-addition of the hydroxyl group of the Thr to the α,βunsaturated carboxamide moiety of SylA (12). SylA preferentially inhibits the proteasomal β-5 subunit (chymotrypsin-like; CT-L) activity with a Ki value of 0.843 µM, weakly inhibits the β2-(trypsin-like; T-L) subunit activity with a Ki value of 6.7 µM, and has no effects on the β1-(caspase-like; C-L) subunit activity (12,15).
We previously synthesized and evaluated a number of SylA-analogs (13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and three other groups have designed syringolin variants (23)(24)(25)(26). In this study, we synthesized TIR-199, one of the most potent SylA-derived compounds to date. We 2 demonstrate that TIR-199 inhibits the proteasome activity and impedes MM and other tumor cell growth, with a significantly higher potency than the natural product SylA, both in vitro and in vivo. Importantly, this represents the first study to examine the efficacy of a syrbactin in animals.

Total synthesis of Syrbactin Structural
Analog  and Reagents -The synthetic route to TIR-199 (MW 534) is provided in Scheme 1 and described in detail in the Supplemental Methods. Most of the route follows our reported syringolin A synthesis (22), with the exception of the use of alaninol. From a (S)-vinylglycine starting material, the route entails 10 steps and proceeds in 10% overall yield. Stocks were prepared in DMSO (10 mM). The natural product syringolin (SylA) and bortezomib (BTZ) served as controls throughout the study and were dissolved in sterile water and DMSO, respectively.
Computational Assessment of Selectivity -We used computational modeling and energetic analysis of the covalently bound complexes of TIR-199 to each proteasome receptor to understand the selectivity of TIR-199 to the catalytic receptor subtypes of the human proteasome. Because no experimental structure is available for the human proteasome, we performed homology modeling to derive the human proteasome atomic structure from the X-ray crystallographic data of the Bos taurus proteasome (PDB id: 1IRU), which has a sequence similarity of 99.6% to that of Homo sapiens. Molecular recognition takes place within a distinct binding pocket at each catalytic site in the proteasome, which includes the active catalytic enzyme and the domain directly adjacent to the active site ( Fig.  2A). Therefore, three binding pocket structures were constructed from the homology model for use in all subsequent calculations, simulations, and analyses: The C-L (catalytic β1 and neighboring β2), T-L (catalytic β2 and neighboring β3), and CT-L (catalytic β5 and neighboring β6) binding pockets. AutoDock 4.2 was used to determine low-energy conformational states of TIR-199 within each of the catalytic receptors when bound covalently to the catalytic Thr1 active site (27). AMBER version 11 was subsequently used to perform molecular dynamics simulations starting with the bound-state conformations obtained from docking (28). The AMBER GAFF parameters were applied to the novel ligand TIR-199, while the AMBER 99SB protein forcefield parameterized the protein structures. Covalent bonds were manually formed using the AMBER xleap utility, and parameters bridging the GAFF and 99SB systems were derived from the GAFF forcefield (29). Implicit solvent Langevin dynamics simulated all systems. Ten thousand equilibration steps were performed at 100K, 200K, and 298K. Production simulations of each system were performed for 10 nanoseconds simulation time at 298K on the XSEDE national super computer system. For all simulations, a selection of protein atoms within 10Å of the ligand's initial positions defined the unconstrained, mobile region of the simulation, while the rest of the protein structure was held fixed. Trajectories of the molecular dynamics simulations were subject to molecular mechanics/generalized Born/surface area (MMGBSA) energetic postsimulation analysis to calculate accurate ligandreceptor affinities over each recorded snapshot along the simulation trajectories (30,31). The total MMGBSA interaction energy was calculated for every frame in each trajectory. The energies were separated into polar interactions, combining Coulombic and generalized Born interaction energies, and nonpolar interactions, combining van der Waals and non-polar solvation solvent-accessible surface area (SASA) interaction energies.
In Vitro Proteasome Activity Assay -To determine the anti-proteasome activity of TIR-199 in the in vitro environment, we measured the three catalytic activities (β1, β2, β5) as previously described (12,13,15). This assay uses purified 20S constitutive proteasome from human erythrocytes or immunoproteasome from human peripheral blood mononuclear cells (PBMCs) (Enzo Life Sciences) and luminogenic substrates (Z-LRR-Glo™, Z-nLPnLD-Glo™, and Suc-LLVY-Glo™), specific for the three β1, β2, and β5 catalytic subunit activities (also referred to T-L, C-L, and CT-L activities, respectively). The 20S proteasome and the specific luminogenic substrates were incubated individually with compounds at increasing concentrations (0 to 10 μM). Controls included the natural product SylA (20 μM) and BTZ (0.01 μM). Following cleavage by the 20S proteasome, the substrate for luciferase (aminoluciferin) is released, allowing the luciferase reaction to produce light. The luminescence was recorded as relative light unites (RLU) after 30 min incubation on a 96-well microplate luminometer.
In Vivo Proteasome Activity Assay -To determine the anti-proteasome activity of TIR-199 in the culture environment, the proteasome-Glo inhibition assay was performed as previously described (13,21). Solid white 96-well microtiter cell culture plates were seeded with cells and proteasome inhibition was measured using the proteasome Glo™ reagent according to the manufacturer's instructions (Promega). In brief, MM1.RL or MYCN2 cells were treated with TIR-199 at different concentrations (0-10 µM) as indicated and incubated for 2 hrs, followed by incubation for 10 min with 100 µl of proteasome Glo reagent, containing the bioluminescent substrates Suc-LLVY-aminoluciferin, Z-nLPnLD-aminoluciferin, and Z-LRRaminoluciferin were added to measure the CT-L, C-L, and T-L activities, respectively. Luminescence was measured with a Multi-Mode Synergy™ MX Microplate Reader (BioTek, Inc.) and expressed as relative light units (RLU). SylA and BTZ were used as controls.
In Vivo Proteasome Sensor Assay -The proteasome sensor activity assay was performed in HEK-293 cells according to the manufacturer's instructions (Clontech). ZsProSensor-1 is a proteasome-sensitive fluorescent reporter, producing a fusion of green fluorescent protein (GFP) with a degradation domain (ODC) that targets the protein for rapid degradation by the proteasome. As a consequence, green fluorescence accumulates in those cells in which the proteasome is inhibited. Unlike the previous cell culture-based assay, this test does not require exogenous addition of bioluminescent substrates as it expresses an internal probe. Cells were transfected with the ZsProSensor-1 vector and tested in the presence of increasing concentrations (0-1 µM) of TIR-199 or BTZ for 24 hrs. GFP expression was quantified using an ELISA reader and pictures taken with an immunofluorescence microscope.
Mammalian Cell Cultures -The multiple myeloma (MM) cell line MM1.RL is derived from the parent cell line MM1, established from peripheral blood of a multiple myeloma patient with acquired resistance to steroid-based (dexamethasone) therapy, and was kindly provided by N. Krett (Northwestern University) (32). The human neuroblastoma (NB) cell line MYCN2 was derived from parent cell line SH-EP, and was kindly provided by Dr. J. Shohet (Texas Children's Hospital) (33). Human embryonic kidney 293 (HEK-293) cells were from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HepG2 cells were tested at Cerep, Inc. (Redmond, WA, USA). Cell lines were maintained in RPMI 1640 medium (MM1.RL, MYCN2), DMEM medium (HEK-293) (Mediatech Inc., Manassas, VA, USA) or MEM alpha growth medium (HepG2). RPMI 1640 and DMEM medium contained 10 % (v/v) heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 and seeded 16-24 hrs prior to the start of the assay. 4 Cell Viability Assay -The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay is a homogeneous, colorimetric method for determining the number of viable cells in cell culture assays. The assay is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent [phenazine methosulfate; PMS] (Promega, San Luis Obispo, CA, USA). MTS is bioreduced into soluble formazan product by dehydrogenase enzymes found in metabolically active cells. The viability of cancer cells was determined after 24 hrs treatment with TIR-199 at indicated concentrations [0-10 µM] by measuring the absorbance of the formazan product at 490 nm using a Multi-Mode Synergy™ MX Microplate Reader (BioTek, Inc., Winooski, VT, USA) as previously described (14). Data were expressed in percent (%) cell viability relative to untreated control cells. SylA and BTZ were used as positive controls.

NCI-60 Human Tumor Cell Line Screen
-The NCI-60 cell line panel includes 60 human tumor cell lines. The effect of SylA and TIR-199 on tumor cell growth was tested at the National Cancer Institute, Developmental Therapeutics Program (NCI-DTP), according to their standard protocols and as previously published (34). For additional information, see: https://dtp.cancer.gov/discovery_development/ nci-60/methodology.htm.
ADR Profiling -The adverse drug reaction (ADR) profile was performed at Cerep, Inc. Many ADRs are linked to off-target activities at a great variety of cellular receptors and enzymes. The effect of TIR-199 (10 μM) against the kidney organ system was tested by measuring 17 molecular targets selected on the basis of known associations of individual targets with serious ADRs as well as statistical associations derived from Cerep's proprietary BioPrint®. The following receptors/targets were individually tested: 5-HT2A, 5-HT2C, alpha2B adrenergic, beta1 adrenergic, dopamine 2S, muscarinic acetylcholine (M2, M4, M5), neurokinin 2, urotensin, and parathyroid hormone 1 receptor. In addition, activation of adenylate cyclase C and inhibition of enzymes COX2, ACE, dipeptidyl peptidase IV, phospholipase C, acetylcholinesterase was measured. The binding, activation or inhibition for each target at greater than 50% compared to control was considered a significant effect.
In Vitro Cytotoxicity Assay -This high content cytotoxicity assay was performed at Cerep, Inc. Five end points were simultaneously measured in individual cells, avoiding the drawbacks of classic in vitro cytotoxicity assays, which measure nonspecific and late occurring cytotoxic events. The five end points are cellular parameters such as mitochondrial membrane potential (TMRM) and intracellular free calcium (Fluo-4) and more classic parameters such as nuclear size (Hoechst), membrane permeability (TOTO-3), and cell number (Hoechst), in live HepG2 cells. In brief, HepG2 cells (passage 1-15) were plated in 96-well poly D-lysine-coated plates at 3,000 cells/well in MEM alpha growth medium 16-24 hrs prior to start of the assay. One hour prior to the addition of test compound, the cells were equilibrated with assay medium containing 1% FBS. Cells were treated with TIR-199 (100 nM) or BTZ (5 nM) for 72 hrs. At the end of the incubation period, the cells were loaded for one hour with a dye cocktail containing Hoechst, TMRM, Fluo-4, and TOTO-3. Plates were scanned with an automated fluorescent microscope. Image analysis software was used to quantitate surface area (nuclear size) and fluorescence intensity (rest of end points) in defined cellular areas. Data were normalized and expressed as % of effect relative to the untreated controls. Data were also normalized using a reference compound (cerivastatin), whose maximum effect at any concentration was considered the 100% effect.
In Vivo Acute Toxicity Study -The maximum tolerated dose (MTD) study was performed at the NCI-DTP. To investigate acute toxicity of TIR-199 in vivo, the MTD was determined in female athymic nude mice. TIR-199 was given by intraperitoneal (IP) injection QD x 1 on Day 0, at 12.5, 25.0, and 50.0 mg/kg/dose (groups 1-3; n=1/group). Injection volume was 0.05, 0.1 or 0.2 ml/10 gm body weight (2.5 mg/ml, homogeneous smooth suspension in 10% DMSO in Saline/Tween 80). The mice were held for 14 days postdosing to monitor for delayed toxicity (bone marrow suppression, irreversible liver/kidney and other organ damage). For additional information, see: https://dtp.cancer.gov/organization/btb/acute_to xicity.htm.
In Vivo Hollow Fiber Assay -The in vivo hollow fiber assay was performed at the NCI-DTP. A solid tumor efficacy mouse model based on cell growth inside biocompatible hollow fibers was used to provide quantitative indices of drug efficacy in heterogeneous tumors with minimal time and material expenditures (35). Small hollow fibers (1 mm in diameter, 2 cm long, molecular weight exclusion of 500,000 Da), made of polyvinylidene fluoride and containing cells from human tumors, were inserted underneath the skin and in the body cavity of the mouse. A standard panel of 12 tumor cell lines, including lung, breast, colon, melanoma, ovarian, and central nervous system, was used for the routine hollow fiber screening of in vitro actives. Each mouse received three tumor cell lines as intraperitoneal (IP) implants, and three as subcutaneous (SC) implants. A total of 24 mice were used in 8 groups (n=3/group, each group representing two dose levels with 4 experiments, 3 cell lines/experiment) and treated by IP injection with TIR-199 starting on day 3 or 4 following fiber implantation and continuing daily for four days (QD X 4). TIR-199 was administered at two doses (9.4 mg/kg/dose or 6.3 mg/kg/dose). The selected high and low doses were based on the MTD determined in the in vivo acute toxicity study, using the formula: High dose = [MTD x 1.5]/4 and Low dose=0.67 x high dose. The fibers were collected from the mice on the day following the fourth compound treatment and subjected to the stable endpoint MTT assay.
The optical density of each sample was determined by spectrophotometry at 540 nm, and the mean of each treatment group was calculated. The percent net growth for each cell line in each treatment group is calculated and compared to the percent net growth in the vehicle treated controls. A 50% or greater reduction in percent net growth in the treated samples compared to the vehicle control samples is considered a positive result. Each positive result is given a score of 2, and all of the scores are totaled for a given compound. The maximum possible score for an agent is 96 (12 cell lines × 2 sites × 2 dose levels × 2). A compound is considered effective and of potential interest for further xenograft studies at NCI if it has a combined IP + SC score of ≥ 20. Appropriate controls were included (n=12 for blank fibers, n=24 for vehicle controls, and n=24 for positive controls using paclitaxel). For additional information, see:https://dtp.cancer.gov/organization/btb/holl ow_fiber_assay.htm.
Statistical Analyses -All experiments were performed in three independent experiments (n = 3) unless otherwise stated. Error bars indicate the standard error of the mean (± SEM). Data were prepared using the Microsoft Excel (Redmund, WA, USA) and GraphPad Prism 6 (La Jolla, CA, USA).

Structure and Synthesis of TIR-199 -
The need to improve both the intrinsic potency and physicochemical properties of the syringolins led us to related natural products, the glidobactins, which were discovered based on their activity against tumor cell lines (36,37). They share a 12-membered macrodilactam, but the glidobactins have a much less hydrophilic side chain, and also differ in the amino acid (valine vs. alanine) used biosynthetically to form the α,βunsaturated amide (Fig. 1). Since that grouping is the pharmacophore of both the syringolins and glidobactins that reacts with the essential Thr1 hydroxy group of proteasome β-subunits, 6 we assumed that a less sterically demanding group adjacent to the unsaturated amide could enhance activity. These considerations suggested a number of structural analogs, one of which, TIR-199, proved the most interesting (Fig. 1).
The synthetic route to TIR-199 is provided in Scheme 1 and described in detail in the Supplemental Methods section. Most of the route follows our reported syringolin A synthesis (22), with the exception of the use of alaninol. From a (S)-vinylglycine starting material, the route entails 10 steps and proceeds in 10% overall yield.
Computational Modeling and Docking of TIR-199 Into the Proteasome -Structureactivity relationships of the dozens of natural and synthetic syrbactins were examined to assist understanding the intrinsic activity of TIR-199 against the proteasome ( Fig. 2A). Replacement of the syringolin R 3 with a straight-chain alkane was first reported by Kaiser's group (15), and while expected to significantly improve the cell-based activity by eliminating charge, also benefitted its intrinsic potency. Regarding the change of the larger R 1 of syringolin A to a methyl group, the proteasome's catalytic Thr1 hydroxyl approaches the unsaturated lactam adjacent to this position, making a larger grouping there a significant steric impediment (Fig. 2B). Syringolin A is more potent than its monounsaturated natural relative syringolin B (A-B = CH 2 CH 2 ), and conformational analysis suggests the effect of the second alkene is to introduce strain into the macrodilactam and make it more reactive with the proteasome.
The bound-state affinities for TIR-199 to each active site within the human proteasome were calculated using MM-GBSA analysis of molecular dynamics trajectories. The most favorable interaction was found between TIR-199 and CT-L receptor due to unique non-polar stabilization. With decreasing non-polar affinity, the selectivity continues with the C-L receptor, followed by T-L receptor. The average polar and non-polar potential energies are summarized in Table 1.

TIR-199 Inhibits Catalytic Subunit
Activities of the Proteasome -To measure the effect of TIR-199 on the proteasome, we measured the β1 (C-L), β2 (T-L), and β5 (CT-L) catalytic subunit activities of the proteasome. In vitro experiments with either purified 20S constitutive proteasome from human erythrocytes, or 20S immunoproteasome from human peripheral blood mononuclear cells (PBMCs) showed that TIR-199 strongly inhibited CT-L and T-L, but not C-L activity of the proteasome, in a dose-dependent manner (Fig. 3). Bortezomib (BTZ) was used as a control and inhibited all three constitutive proteasome activities indiscriminately but interestingly, did not inhibit the immunoproteasome T-L activity. TIR-199 displayed significantly improved potencies on the constitutive proteasome CT-L activity compared to SylA (Ki of 0.018 and 0.843 µM (12), respectively), but was less potent than BTZ (Ki < 0.01 µM) (Fig. 3). Interestingly, TIR-199 was less effective against the immunoproteasome CT-L activity (Ki of ~0.075 µM) (Fig. 3B). Table 2 compares the activity of TIR-199 against previously reported syrbactins (SylA, GlbA, and SylA-GlbA hybrid) and bortezomib (12,13,38). The activity of TIR-199 was comparable with the activity of the SylA-GlbA hybrid.
To verify the potency against the proteasome in actively growing cell cultures, we tested TIR-199 in a cell-based, in vivo proteasome activity assay. TIR-199 strongly inhibited the catalytic β1, β2, and β5 subunit activities in MM1.RL cells in a dose-dependent manner (Fig. 4A,C,E). Similar data were obtained with MYCN2 neuroblastoma (NB) cells, confirming the activity of proteasome inhibition in two distinct cancer cell types (Fig.  4B,D,F). Slightly higher doses of TIR-199 were required in NB cells to achieve the effects observed in MM cells. The T-L activity in NB cells was not significantly reduced.
The effect of TIR-199 on the proteasome in actively growing cells was independently confirmed with the proteasome sensor activity assay in which the blockage of proteasome function is reflected by the accumulation of an internally overexpressed substrate (rather than externally-added substrate, as shown in Fig. 4) fused to GFP. TIR-199-treated cells as well as BTZ-treated cells showed strong accumulation of GFP compared to untreated control cells (Fig. 5). This result shows that TIR-199 is able to penetrate the membrane of intact cells and inhibits the proteasome of actively dividing cells.

TIR-199 Induces Cell Death in Multiple
Myeloma and Neuroblastoma Cells -To investigate if TIR-199 induces cancer cell death, we tested increasing TIR-199 concentrations against MM and NB cells. TIR-199 killed the MM cell line MM1.RL very rapidly at the lowest concentration (0.05 µM), and inhibited NB cell viability in a dosedependent manner between 0 and 10 µM (Fig.  4G,H). The concentrations to induce 50% cell death (IC 50 ) were estimated at < 0.05 µM and ~ 0.1 µM for MM and NB cells, respectively. These IC 50 values reflect a more than 250-fold increase in potency compared to SylA that has an IC 50 of 20-25 µM (11) and up to 39 µM in MM1.RL cells (14). The fact that TIR-199 is able to induce strong cell death in the MM1.RL cell line is particularly important and confirms that proteasome inhibition is a successful strategy to treat dexamethasone-resistant MM tumors. Table 3 compares the effect of TIR-199 on cancer cell death against previously reported syrbactins (SylA, GlbA, and SylA-GlbA hybrid) and bortezomib (11,13,14). The activity of TIR-199 was better or comparable to GlbA and SylA-GlbA hybrid.

NCI-60 Human Tumor Cell Line Screen with
TIR-199 -To explore its utility in other cancer cell types, TIR-199 was tested at the National Cancer Institute Development Therapeutics Program (NCI-DTP). The results confirmed that TIR-199 induces dose-dependent cell death in a wide range of tumor cell types including breast, CNS, colon, kidney, lung, ovarian, skin, and prostate cancer, and leukemia. Of note, TIR-199 was active in a panel of kidney tumor cell lines, with exceptionally high activities in four cell lines (RXF 393, TK-10, A498, and SN12C) (Fig. 6). Compared to other groups of the NCI-60 cell panel, the renal cancer panel often does not respond well in drug screens, and, therefore, the results with TIR-199 are particularly encouraging (NCI-DTP, personal communication). TIR-199 also exhibited high activity in other cell lines, including RPMI-8226 (leukemia), NCI-H522 (non-small cell lung cancer), KM12 (colon cancer), SNB-75 (CNS cancer), LOX IMVI (melanoma), OVCAR-3 (ovarian cancer), PC-3 (prostate cancer), and BT-549 (breast cancer). Overall, our data suggest that TIR-199 is a promising anticancer agent with broad application potential against various tumor groups and selective activities in certain cell lines within each tumor group. To further demonstrate the improved anti-tumor cell activity, the initial One Dose Mean Graphs of TIR-199 was compared side-by-side with SylA, clearly showing that TIR-199 has significantly higher activity potential than the natural product SylA (Fig. 7).
Preclinical In Vitro and In Vivo Evaluation of TIR-199 -Drug induced toxicity is one of the major causes of failure during drug development and the major reason for removal of approved drugs from the market. To assess the potential of TIR-199 for preclinical development, a broad range of biological screening tests were performed. Because one potential therapeutic area identified by the NCI for TIR-199 is kidney cancer (Fig. 6), we were interested in an adverse drug reaction (ADR) assessment for kidney targets. The Cerep ADR kidney panel included 17 assays, which determine the binding of TIR-199 (10 µM) to the following targets/receptors: 5-HT2A, 5-HT2C, alpha2B adrenergic, beta1 adrenergic, dopamine 2S, muscarinic acetylcholine (M2, M4, M5), neurokinin 2, urotensin, and parathyroid hormone 1 receptor. In addition, activation of adenylate cyclase C and inhibition of enzymes COX2, ACE, dipeptidyl peptidase IV, phospholipase C, acetylcholinesterase was measured. For the radioligand binding assays to the receptors, the strongest antagonism observed was 8% at the test concentration. Functional assays of parathyroid hormone 1 receptor and adenylate cyclase activation showed no effect of TIR-199. The enzymatic activity most affected by it was dipeptidyl peptidase IV, which was inhibited 26% at the test concentration.
As an effective concentration for proteasome inhibition by TIR-199 is 1000-fold lower than these test concentrations, none of these off-target effects is considered significant (data not shown).
Additional assays were performed to determine the cytotoxicity of TIR-199 in HepG2 cells. Readouts were based on five end points that are measured simultaneously in individual cells: cell number (decreased numbers indicate cell death and/or decreased proliferation), free calcium release (uncontrolled increase in cytoplasmic calcium indicates cellular toxicity), membrane permeability (disruption of the cytoplasmic membrane indicates late cellular toxicity), mitochondrial membrane potential (indicator of respiratory capacity and cellular energetics), and nuclear size (nuclear shrinkage by chromatin condensation indicates apoptotic cell death). TIR-199 (100 nM) was tested and compared in parallel with BTZ (5 nM). The results are summarized in Table 4. Other than in direct cell killing, TIR-199 and BTZ had similar effects on intracellular calcium, nuclear size, membrane permeability, and mitochondrial membrane potential.
Due to the encouraging results of the NCI-60 cell line panel, the NCI-DTP performed in vivo studies in mice to determine the maximum tolerated dose (MTD) and moved TIR-199 forward into in vivo hollow fiber assays. Non-tumored athymic nude female mice were injected (IP) with a single dose of TIR-199 at 12.5, 25, and 50 mg/kg/dose (groups 1-3; n=1/group). At 25 mg/kg treated mice were alive at day 19 while at 50 mg/kg, the mice died on day 2, suggesting an MTD for TIR-199 of 25 mg/kg (Table 5). For comparison, the classical anti-cancer drug paclitaxel (Taxol ® ) is highly active at 15 mg/kg and lethal at a 30 mg/kg (NCI-DTP, personal communication). Importantly, TIR-199 at 9.4 mg/kg/dose and 6.3 mg/kg/dose (total of four doses, IP) was effective in hollow fiber studies in mice using twelve tumor cell lines (hollow fibers implanted either IP or SC) and inhibited the cell proliferation to various degrees in representing tumors of the breast, CNS, colon, lung, ovary, and skin ( Table 6). Most notably, TIR-199 inhibited colon, melanoma, and ovarian tumor cells. Overall, tumor cells in IPimplanted hollow fibers responded more readily to IP-administered TIR-199, with the exception of melanoma tumors, which were also significantly inhibited in SC-implanted hollow fibers. Most but not all tumor cells that were inhibited by TIR-199 in the in vivo hollow fiber assay were also highly responsive in the NCI-60 cell line screen ( Figure 6).

DISCUSSION
Natural products continue to offer attractive lead compounds for novel anticancer therapeutics (39), with the syrbactins as just one recent example. It is notable that the FDAapproved proteasome inhibitor carfilzomib closely resembles the peptide natural product epoxomicin. Likewise, TIR-199 addresses deficits of the original lead, SylA, through reduction of hydrophilicity and enhancement of potency. Its ligand efficiency, a broadly used measure of the potential of a compound for pharmaceutical lead development (40), based on its potency at the proteasome CT-L site is 0.28, within the desirable range.
Our understanding of TIR-199 activity was enhanced through simulation of its interaction with the chemical environment of each receptor when bound to the three catalytic sites of the proteasome. This approach revealed an interaction of the TIR-199 side chain with a proteasome pocket that is absent in any previous drug-proteasome structure (41). A unique pocket behind the binding site of the CT-L receptor led to exceptionally high nonpolar affinity and conformational stability of TIR-199 in the binding site (Fig. 2B). In addition, a unique rotation after the peptide bond was required for the ligand to extend into the deep pocket, lowering the internal energy of the ligand by 2.5 -3 kcal/mol relative to the other starting conformations.
In the in vitro experiments, TIR-199 inhibited the β2-and β5-catalytic subunit activities in a dose-dependent fashion, with highest affinity towards the β5 subunit and only minimal activity towards the β1 subunit. This is similar to what was observed with SylA (12,15). Interestingly, in the cell-based proteasome activity assay, all three activities were inhibited in a similar fashion in MM, but not in NB cells. The reason for these differences is not clear but may in part be due to cell type-specific differences and/or differences in assay conditions (in vitro proteasome activity assay versus a cell-based in vivo proteasome activity assay) and the different sources of luminogenic substrates used in each assay. TIR-199 strongly induced tumor cell death in MM and NB cells but also in a wide range of other tumor cell lines as shown in the NCI-60 cell panel screening results (Fig. 6). Importantly, four of eight cell lines in the kidney cancer panel that traditionally responds poorly to compounds tested in the NCI-DTP drug pipeline were exceptionally sensitive to TIR-199. In the hollow fiber mouse tumor model, TIR-199 was most notably active in colon, melanoma, and ovarian tumors (Table 6). Of note, several leading drugs like bortezomib, paclitaxel, romidepsin, eribulin, sipuleucel-T, and dinutuximab (Ch14.18) were initially studied at the NCI-DTP using hollow fiber assays in mice to obtain quantitative indices of in vivo drug efficacy.
Proteasome inhibitors might be explored for use in diseases unrelated to cancer, in which aberrant regulation of the proteasome or immunoproteasome has been observed. Such diseases include Huntington disease (42), Alzheimer's disease (43), macular degeneration (44), inflammatory bowel disease (Crohn disease, ulcerative colitis) (45,46), and rheumatoid arthritis (Sjogren's syndrome) (47). In some instances, the use of proteasomespecific inhibitors that directly target the catalytic core might useful. For example, the proteasomal CT-L activity is increased in neurosensory retina with disease progression in age-related macular degeneration (44). In Crohn disease and ulcerative colitis proteasome inhibitors may be effective in blocking the proteasome-mediated activation of the NF-ΚB pathway in inflammatory bowel disease patients (45,46). In other instances, inhibitors that alter the expression of immunoproteasomespecific subunits might lead to novel treatment options. For example, the immunoproteasomespecific subunits LMP2 or LMP7 are increased in Huntington disease neurodegeneration (42) and tissue-specific upregulation of LMP7 is characteristic in patients with Sjogren's syndrome (47).
In conclusion, this is the first study that tests a syrbactin-related proteasome inhibitor in animals. We demonstrate that TIR-199 has broad-range anti-tumor activity in vivo. Therefore, TIR-199 and novel TIR-199-derived analogs bear significant potential for further preclinical development into a clinical drug to treat MM, kidney, and other cancer forms.
CONACYT (to T.R.I-R). Contributions via experiment.com from Francois Souchay, Yong Rok Lee, Bruce Liu, Ann Adenbaum & Alan Schramm, and Robert Patton are also appreciated. We are most thankful to the National Cancer Institute Developmental Therapeutics Program (NCI-DTP) for performing the NCI-60 human tumor cell line screen, in vivo acute toxicity study, and the in vivo hollow fiber assays with TIR-199. We thank Dr. Paul Grothaus (NCI-DTP) for his support and for insightful discussion of results. We are grateful to Nancy L. Krett (Northwestern University, Chicago, IL, USA) and Jason Shohet (Texas Children's Hospital, Houston, TX, USA) for providing multiple myeloma cell line MM1.RL and neuroblastoma cell line MYCN2, respectively. We are indebted to Dr. Dirk Geerts (Rotterdam, the Netherlands) for excellent scientific discussion and for editing the manuscript.  A unique pocket exposed to the surface of the proteasome complex is visible behind TIR-199. Extension of the hydrophobic tail into this pocket leads to increased affinity and stability.     -response curves for TIR-199 (NSC:761526). A, A panel of 60 human tumor cell lines representing nine different cancerous tissues of origin (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer) was tested at the National Cancer Institute Developmental Therapeutics Program (NCI-DTP) in the presence of TIR-199 at five concentrations. TIR-199 was tested over a 10,000-fold concentration range in a 2-day assay and exhibited dose-dependent growth inhibition to various degrees, in all tested tumor cell lines. B, Dose-response data of (A) were used to calculate three endpoints for each cell line -GI50 (the Log 10 of the concentration that caused 50% growth inhibition), TGI (the Log 10 of the concentration that caused total growth inhibition), and LC50 (the Log 10 of the concentration that caused 50% lethality). For each endpoint the mean across all the cell lines was calculated. The GI50 data are graphed as the difference of the GI50 for a particular cell line from the mean GI50. Cell lines that are more sensitive are represented as bars deflecting to the right of the mean and less sensitive cell lines project to the left of the mean. TGI and LC50 Mean Graphs are generated in a similar fashion. One representative data set of three independent experiments is shown (n=3), except for the leukemia cell panel (n=2). For additional information about the NCI-60 cell line panel, see [34] and Experimental Procedures.     Table 3. Effect of TIR-199 and other syrbactins on cancer cell death. Bortezomib was used as control for comparison. a IC 50 = inhibitory concentration at which cell viability is reduced by 50%. *Instead of SK-N-SH, the MYCN2 cell line was used in experiments with TIR-199. MYCN2 represents the neuroblastoma cell line SHEP which was previoulsy transfected with a tetracycline on/off plasmid system to allow for controlled MYCN expression (33).   Table 4. Values represent % effect (% cytotoxicity) relative to untreated controls. This includes -cell number: % reduction; intracellular free calcium: % increase; nuclear size: % reduction; membrane permeability: % increase; mitochondrial membrane potential: % reduction. Data represent the mean of triplicate wells (n=3).