Stefan Proniuk

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication

We have recently demonstrated that AR‐12 (OSU‐03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR‐12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR‐12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α—dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over‐expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR‐12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR‐12—stimulated the co‐localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug‐induced autophagosome formation and reduced the anti‐viral protection afforded by AR‐12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR‐12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR‐12 acts as a broad‐specificity anti‐viral drug in vitro and in vivo. We argue future patient studies with AR‐12 are warranted. J. Cell. Physiol. 231: 2286–2302, 2016.

The celecoxib derivatives AR-12 and AR-14 induce autophagy and clear prion-infected cells from prions

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR-12 has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a potential therapeutic agent for neurodegenerative disorders. In this study, we investigated the role of AR-12 and its derivatives in controlling prion infection. We tested AR-12 in prion infected neuronal and non-neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR-12 and its analogue AR-14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 or AR-14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro and in vivo. Taken together, this study demonstrates that AR-12 and the AR-14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

AR-12 Inhibits Chaperone Proteins Preventing Virus Replication and the Accumulation of Toxic Misfolded Proteins

Laurence Booth1, Jane L Roberts1, Heath Ecroyd2, St. Patrick Reid3, Stefan Proniuk4, Alexander Zukiwski4, Abraham Jacob5, Elsa Damonte6, María J Tuñón7 and Paul Dent1* 1Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA 2School of Biological Sciences and Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia 3Molecular and Translational Science, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 21702-5011, USA

Abstract 4523: Population pharmacokinetic (PPK) modeling of onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers

Background: Onapristone is a type I PR antagonist, which prevents PR-induced DNA transcription. Onapristone anti-cancer activity is well documented. An extended-release (ER) tablet formulation of onapristone was designed to address the liver function test (LFT) elevations seen with immediate-release (IR) onapristone. A phase 1 study with onapristone in patients with tumors expressing PR is underway. Objectives included determining the PK profile of ER onapristone using a PPK approach. Materials and methods: This is an ongoing multi-center, open-label, randomized, parallel-group, 2-stage ph1 study. Female pts ≥18 yrs with tumors expressing PR are eligible. The Stage 1 primary endpoint is the recommended ph2 dose of ER onapristone; secondary endpoints include: safety, efficacy, and PK. Pts received onapristone ER 10, 20, 30, 40 or 50 mg BID, or onapristone IR tablets 100 mg QD until progressive disease or intolerability. PK blood samples from 8 time points were collected over 12 h post-dose Day 1 for the ER and 9 blood samples over 24 h post-dose for the IR formulation. Onapristone plasma concentrations were measured using validated UPLC with tandem mass spectrometry detection (range 1-250 ng/mL). Monolix V4.1 was used to calculate absorption constant (K a ); apparent clearance (CL/F); inter-compartmental clearance (Q); apparent distribution volume (V1/F), 2 nd compartment distribution volume (V2) and bioavailability (F) of ER vs IR. Results: Stage 1 is complete. 42 pts have validated PK data. A 2-compartment open model adequately described the total onapristone time-concentration curve with linear elimination. Results are in Table 1. Conclusions: The PPK modeling described the plasma onapristone time-concentration curves well. A central volume equivalent to the circulating blood volume and a large volume of the deep compartment suggest a large tissue diffusion. PPK/PD modeling to explore safety and efficacy is ongoing, with no overt PK/safety relationship detected. Citation Format: Keyvan Rezai, Paul Cottu, Samuel Huguet, Mario Campone, Antoine Italiano, Andrea Varga, Jacques Bonneterre, Alexandra Leary, Marie-Paule Sablin, Stefan Proniuk, Alice Bexon, Erard Gilles, Joseph Bisaha, Alexander Zukiwski, Francois Lokiec. Population pharmacokinetic (PPK) modeling of onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2015-4523

Phase I study of onapristone, a type I antiprogestin, in female patients with previously treated recurrent or metastatic progesterone receptor-expressing cancers

Introduction Onapristone is a type I progesterone receptor (PR) antagonist, which prevents PR- mediated DNA transcription. Onapristone is active in multiple preclinical models and two prior studies demonstrated promising activity in patients with breast cancer. We conducted a study of extended release (ER) Onapristone to determine a recommended dose and explore the role of transcriptionally-activated PR (APR), detected as an aggregated subnuclear distribution pattern, as a predictive biomarker. Methods An open-label, multicenter, randomized, parallel-group, phase 1 study (target n = 60; NCT02052128) included female patients ≥18 years with PRpos tumors. APR analysis was performed on archival tumor tissue. Patients were randomized to five cohorts of extended release (ER) onapristone tablets 10, 20, 30, 40 or 50 mg BID, or immediate release 100 mg QD until progressive disease or intolerability. Primary endpoint was to identify the recommended phase 2 dose. Secondary endpoints included safety, clinical benefit and pharmacokinetics. Results The phase 1 dose escalation component of the study is complete (n = 52). Tumor diagnosis included: endometrial carcinoma 12; breast cancer 20; ovarian cancer 13; other 7. Median age was 64 (36–84). No dose limiting toxicity was observed with reported liver function test elevation related only to liver metastases. The RP2D was 50 mg ER BID. Median therapy duration was 8 weeks (range 2–44), and 9 patients had clinical benefit ≥24 weeks, including 2 patients with APRpos endometrial carcinoma. Conclusion Clinical benefit with excellent tolerance was seen in heavily pretreated patients with endometrial, ovarian and breast cancer. The data support the development of Onapristone in endometrial endometrioid cancer. Onapristone should also be evaluated in ovarian and breast cancers along with APR immunohistochemistry validation.

Exploring the in vitro potential of celecoxib derivative AR-12 as an effective antiviral compound against four dengue virus serotypes

Objectives: With no clinically effective antiviral options available, infections and fatalities associated with dengue virus (DENV) have reached an alarming level worldwide. We have designed this study to evaluate the efficacy of the celecoxib derivative AR‐12 against the in vitro replication of all four DENV serotypes. Methods: Each 24‐well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR‐12. Following 48 h of incubation, inhibitory efficacies of AR‐12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT‐PCR. The underlying mechanism(s) possibly involved in the compounds inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains. Results: The qRT‐PCR data demonstrated that DENV‐3 was most potently inhibited by AR‐12, followed by DENV‐1, DENV‐2 and DENV‐4. Our molecular docking findings suggested that AR‐12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins. Conclusions: These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR‐12 as a potential therapeutic option for DENV infection.

Abstract 1646: Synthesis of [11C]onapristone for clinical investigation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Onapristone is a type I anti-progestin, which prevents the progesterone receptor (PR) monomers from dimerizing, inhibits ligand-induced phosphorylation and prevents association of the PR with its co-activators, thus preventing PR-induced transcription. Onapristone has the potential to treat patients with endometrial cancer, breast cancer, uterine sarcomas, a potential subset of ovarian cancer, prostate cancer and other tumors in which the progesterone receptor plays a role in growth, proliferation and metastasis. [11C]-radiolabeled onapristone and its visualization via PET-scan, coupled with pharmacokinetic (PK) studies, has the potential to determine tissue-specific and blood PK parameters including tumor/tissue and plasma concentrations, whole body distribution and half-life of onapristone. The aim of this study is the development of a rapid Good Manufacturing Product (GMP) synthesis of parentally administered [11C]onapristone. Methods: The production of [11C]-labeled radiopharmaceuticals used a Tracerlab® FX c-Pro (GEMS) synthesis module. Carbon-11 was produced at Institut Curie-Hopital Rene Huguenin via the 14N(p,α)11C nuclear reaction using a PETTrace cyclotron (GEMS) equipped with a carbon-11 target. Carbon-11 is delivered from the cyclotron as [11C]CO2 in the synthesis module, and reduced to [11C]CH4. Methane is halogenated to [11C]CH3I and converted to [11C]methyl triflate (CH3OTf). To prepare [11C]onapristone, the module was loaded with 1mg of N-desmethyl-onapristone (Arno Therapeutics) and 500µL of DMSO in the reaction vessel (Sigma®). [11C]CH3OTf was bubbled into the reaction vessel and heated at +50°C for 10 minutes. The reaction mixture was then diluted with 1 ml of mobile phase and purified using a semi-preparative HPLC column: Sunfire C18 5µm 250X10mm (Waters®), mobile phase: acetonitrile (Sigma®)/water (Waters®) 50/50 v/v; flow rate 4 mL/min. The product fraction was collected in 40mL of sterile water and was passed through a C18 SEP PAK (Waters®). This fraction was eluted with isotonic saline (Braun®) and ethanol (Sigma®). The resulting formulation was passed through a 0.22µm sterilizing filter into a sterile dose vial. Results: 10 tests were performed. Time of synthesis was 50 minutes. The amount of carbon-11 delivered was 12-14GBq for the irradiation parameters as follows: 10 min; 10µA. The decay-corrected yield of reducing [11C]CO2 to [11C]CH4 was >99%. The yields of preparing [11C]CH3I were 27-29%. Time retention of [11C]onapristone was 8.5-9.5 minutes and the non-decay-corrected radiolabelling yields were 1-2%. Conclusions: We have successfully developed a fully-automated production of [11C]onapristone ready for use in clinical trials. Citation Format: Olivier Madar, Julien Fouque, Stefan Proniuk, Keyvan Rezai, Samuel Huguet, Alexander Zukiwski, Erard M. Gilles, Alice S. Bexon, Francois Lokiec. Synthesis of [11C]onapristone for clinical investigation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2014-1646

Abstract 4636: Pharmacokinetic (PK) food effect study of immediate-release onapristone and its primary metabolite (M1) in healthy female subjects: implications for design of a new formulation

Introduction: Onapristone is an antiprogestin with demonstrated clinical activity in breast cancer, reported to have a t 1/2 between 2 and 4 hours. Drugs such as megestrol acetate and abiraterone generally show variability in absorption and, depending on the formulation, food effect (≥10 x C max variation; ≥5 x AUC variation). A study was conducted to determine the best formulation of onapristone to minimize this variability. Methods: The aim of this study was to determine the pharmacokinetic profile of onapristone and mono-desmethyl onapristone (M1), with and without food. Using a two-period, two-sequence, random assignment cross-over design, twelve healthy female subjects were given 10 mg of an oral immediate release formulation of onapristone either after an overnight fast, or within 30 minutes after a high-fat high-calorie meal, with a 2 week washout between dosing periods. PK sampling was performed following each oral administration at: 0, 15, 30, 45 and 60 minutes, 2, 4, 6, 8, 12, 16 and 24 hours (h). Parameters were calculated using the linear trapezoidal method. Population PK modeling was conducted using the nonlinear mixed effect model. Results: Onapristone plasma t 1/2 (mean ± SD) was 4.36 ±0.81 h for the fasted and 3.76 ±0.36 h for the fed state. The absorption phase appeared linear. Onapristone t max was delayed from 1 to 4 h after food intake. A small effect on the single dose onapristone pharmacokinetic profile was also observed, with a slightly decreased mean C max (18%) and increased AUC 0-last (11%). Mean population CL of onapristone was 5.02 ±0.67 L/h and 6.63 ±0.87 L/h for fasted and fed states respectively. There was no food effect on M1 exposure but a decrease in M1 plasma peaks (∼35%) after food intake. The onapristone time-concentration curve was adequately described by a 2-compartment open model with linear elimination using a population PK approach. A significant food effect (p Conclusion: Clinically meaningful C max can be reached with a 10 mg onapristone dose. The results are consistent with prior observations, indicating that food delayed absorption and increased AUC when taken concomitantly. Food effect is minimal compared to other drugs in class, but onapristone should preferably be administered 2 hours before or 1 hour after meals. This food effect is unlikely to have clinical consequences. As absorption is linear, a sustained release formulation would probably reduce C max by ∼25% while not substantially modifying AUC. Given the estimated t 1/2 of 2-4 hours, adequate concentrations should be maintained with twice a day dosing. Citation Format: Keyvan Rezai, Stefan Proniuk, Alex Zukiwski, Erard Gilles, Didier Chassard, Caroline Denot, Haydee L. Ramos, Alice S. Bexon, Francois Lokiec. Pharmacokinetic (PK) food effect study of immediate-release onapristone and its primary metabolite (M1) in healthy female subjects: implications for design of a new formulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4636. doi:10.1158/1538-7445.AM2014-4636