Zoran Filipovic

Stapled α−helical peptide drug development: A potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy

Significance Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we describe the development of a stapled α−helical peptide lead molecule for the treatment of cancers that possess the intact p53 tumor suppressor protein but are resistant to drug therapy because of the overexpression of inhibitory proteins MDM2 and MDMX. The molecule ATSP-7041 is a highly potent dual inhibitor of both MDM2 and MDMX that is shown to effectively reactivate the p53 tumor suppressor pathway in a mechanism-dependent manner in p53-positive cancers in vitro and in vivo. Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein–protein interaction and may offer a viable modality for cancer therapy.

Discovery of RG7388, a Potent and Selective p53–MDM2 Inhibitor in Clinical Development

Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.

MDM2 Small-Molecule Antagonist RG7112 Activates p53 Signaling and Regresses Human Tumors in Preclinical Cancer Models

MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.

Discovery of Potent and Orally Active p53-MDM2 Inhibitors RO5353 and RO2468 for Potential Clinical Development

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.

Preclinical Optimization of MDM2 Antagonist Scheduling for Cancer Treatment by Using a Model-Based Approach

Purpose: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. Experimental Design: A pharmacokinetic–pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. Results: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. Conclusion: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing. Clin Cancer Res; 20(14); 3742–52. ©2014 AACR.

Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy

The field of small-molecule inhibitors of protein-protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

Abstract 4727: Antitumor activity of the MDM2 antagonist, RG7112

The p53 tumor suppressor is a transcription factor which plays a central role in prevention of tumor development by inducing cell cycle arrest or apoptosis in response to diverse stresses. In unstressed cells, p53 level is tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers as a mechanism to impair p53 function. Antagonists of p53-MDM2 interaction could reactivate p53 and may offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the Nutlins, provided preclinical proof-of-concept for MDM2 antagonists as a new therapeutic option for patients with tumors expressing wild-type p53. RG7112 is a member of the Nutlin family of MDM2 antagonists with improved potency, selectivity and pharmacological properties which is currently in Ph1 clinical evaluation. Here, we describe its biological activity in models of human cancer in vitro and in vivo. RG7112 binds to MDM2 with high affinity in vitro (KD ∼11 nM) and effectively blocks its interaction with p53. Crystal structures of RG7112-MDM2 complexes revealed that this imidazoline molecule interact with the p53-binding pocket of MDM2 by projecting functional groups that mimic critical amino acid residues from the p53 molecule. Treatment of cancer cells expressing wild-type p53 with RG7112 stabilized p53 and activated the p53 pathway as revealed by the induction of multiple p53-regulated genes. This led to cell cycle arrest in G1 and G2 phases followed by apoptosis. It showed potent antitumor activity in vitro against a panel of solid tumor and leukemia cells expressing wild-type p53 with EC50 in the 0.2 - 2.2 µM range. The overall selectivity between the panels of seven mutant and fifteen wild-type p53 lines, expressed as fold difference in the average EC 50 values was 14-fold. As previously shown with nutlin-3, RG7112 demonstrated variable apoptotic activity in a panel of solid tumor cells with the best apoptotic response in cells with mdm2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to nude mice bearing established human tumor xenografts caused dose-dependent tumor inhibition and tumor regression at non-toxic concentration. The in vivo mode of action was confirmed by pharmacodynamic markers, indicating p53 pathway activation and induction of growth arrest and apoptosis in tumor tissues. Our results showed that RG7112 is a potent p53 activator and antitumor agent in vitro and in vivo that may have therapeutic utility in the treatment of solid tumors and leukemia with wild-type p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4727. doi:1538-7445.AM2012-4727

Abstract A156: Preclinical activity of MDM2 antagonist RO6839921, a pegylated prodrug for intravenous administration

The p53 tumor suppressor is a transcription factor that inhibits tumorigenesis by inducing cell cycle arrest or apoptosis in response to diverse stresses. In normal cells, p53 levels are tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers, thereby impairing p53 function. Antagonists of p53-MDM2 interaction can enhance p53 activity and offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the nutlins, provided preclinical proof-of-concept for MDM2 antagonists as therapeutics for patients with tumors expressing wild-type p53. The nutlin family member idasanutlin (RG7388, RO5503781) is an oral small molecule inhibitor of MDM2 currently in clinical testing. Here we describe RO6839921, a pegylated prodrug formulated for intravenous administration. This IV MDM2 antagonist has been developed in order to improve variability in exposure seen with the oral compound, and to allow expansion into indications where patients cannot swallow or absorb the oral idasanutlin. RO6839921 is rapidly metabolized to the active principle (AP) idasanutlin which then binds selectively to the p53 site on the surface of the MDM2 molecule. In vitro testing with the AP shows high affinity with effective displacement of p53 from MDM2, leading to stabilization and accumulation of p53 protein and activation of the p53 pathway. Studies focused on in vivo investigation of activity of the prodrug RO6839921 since esterase cleavage is required to release the AP. The anti-tumor activity of RO6839921 was investigated in several sarcoma xenograft models including highly responsive wild-type (WT) p53, MDM2 overexpressing osteosarcoma models SJSA-1 and MHM. Sustained survival was seen in the WT p53 HT1080 fibrosarcoma model when combined with Doxil. Activity was also seen in the WT p53 MOLM-13 disseminated AML model alone and in combination with cytarabine, in the CRPC model 22rv1, and in the ER+ BCa model MCF-7. In these studies we see an increase in dose commensurate with exposure and activity (prodrug vs oral), less variability, and potentiated activity in combination with relevant therapeutics. Clinical studies with the oral AP (idasanutlin) have shown that p53 may be activated by this novel therapeutic strategy that releases p53 from MDM2 inhibition. In particular, patients with AML exhibit significant clinical activity (ASH 2014). In view of the existing unmet medical need in advanced cancers, and the promising activity seen with idasanutlin, RO6839921 is believed to be a promising agent that may offer new therapeutic options, and is therefore currently in clinical testing in both solid and hematologic malignancies. Citation Format: Brian Higgins, Christian Tovar, Kelli Glen, Aruna Railkar, Zoran Filipovic, Farooq Qureshi, Binh Vu, George Ehrlich, Dan Fishlock, Lin-Chi Chen, Steven Middleton, Gwen Nichols, Kathryn Packman, Lyubomir Vassilev. Preclinical activity of MDM2 antagonist RO6839921, a pegylated prodrug for intravenous administration. [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 A156.

Abstract B55: Antitumor activity of the MDM2 antagonist RG7388.

The p53 tumor suppressor is a transcription factor that inhibits tumor development by inducing cell cycle arrest or apoptosis in response to diverse stresses. In normal cells, p53 levels are tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers, thereby impairing p53 function. Antagonists of p53-MDM2 interaction can reactivate p53 and offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the nutlins, provided preclinical proof-of-concept for MDM2 antagonists as therapeutics for patients with tumors expressing wild-type p53. Subsequently, a member of the nutlin family, RG7112, was the first small molecule inhibitor of MDM2 to be tested clinically. Here we describe, RG7388, a highly optimized agent with analogous mechanism of action representing an entirely new branch of the nutlin family of MDM2 antagonists. Like RG7112, RG7388 binds selectively to the p53 site on the surface of the MDM2 molecule, effectively displacing p53 from MDM2, and leading to p53 stabilization and activation of the p53 pathway. However, RG7388 is derived from a distinct chemical series that binds with higher potency and selectivity to the MDM2 protein. RG7388 has substantially improved pharmacological properties, resulting in superior preclinical efficacy at lower doses and exposures as compared with its predecessor, RG7112. RG7388 elicits growth arrest and apoptosis at approximately 10-fold lower concentrations in vitro and in vivo, has an improved CYP inhibition profile, and a 2.5- to 20-fold lower projected human efficacious dose as compared with RG7112. Tumors with functional p53 signaling and MDM2 over-expression or amplification are likely to be the most sensitive to RG7388, however preclinical studies indicate that tumors with normal MDM2 levels may also respond to this novel therapeutic strategy. Results from preclinical safety and toxicology studies support further exploration of this compound in cancer patients. RG7388 is a promising agent that may offer a new therapeutic option and is currently in clinical testing in both solid and hematologic malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B55. Citation Format: Brian Higgins, Christian Tovar, Kelli Glenn, Antje Walz, Zoran Filipovic, Yu-E Zhang, Markus Dangl, Bradford Graves, Lyubomir Vassilev, Kathryn Packman. Antitumor activity of the MDM2 antagonist RG7388. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B55.

Abstract 4827: Humanized hepsin neutralizing antibody RO5486055 inhibits tumor growth and leads to accumulation of hepsin substrate laminin-332

Hepsin is overexpressed in prostate and other cancers where it is implicated in promoting tumor growth, invasion, and metastasis. To further our understanding of the role of hepsin in prostate and breast cancer, a fully humanized monoclonal antibody that recognizes human and cynomolgus hepsin has been developed. RO5486055 selectively binds to hepsin but not related type II transmembrane serine proteases, and neutralizes hepsin serine protease activity with an IC 50 in the single digit nM range. In LNCaP prostate cancer cells, both shRNA knockdown of the hepsin gene and RO5486055 treatment cause a similar accumulation of the β3 chain of laminin-332, a known hepsin substrate. With ip administration in mice, RO5486055 demonstrates dose-dependent exposure and a long serum half-life of 168-406 hours. RO5486055 attenuates tumor growth in the LNCaP prostate cancer and T-47D breast cancer xenograft models, but not in the CRW-22Rv1 prostate or MCF-7 breast cancer xenografts. In these models, it has been shown that the level of the β3 chain of laminin-332 detected by Western blot analysis predicts sensitivity to RO5486055-mediated growth inhibition. Moreover, accumulation of the β3 chain during RO5486055 treatment correlates with antitumor activity. In 8/10 hepsin-expressing patient-derived prostate tumors, an inverse correlation between hepsin and β3 chain expression is observed. The β3 chain of laminin-332 may therefore be useful as both a predictive and response biomarker for anti-hepsin therapy. RO5486055-mediated tumor growth inhibition is enhanced by combination with the EGFR-targeted antibody cetuximab in LNCaP prostate cancer xenografts, and by combination with hormone withdrawal in T-47D breast cancer xenografts. These preclinical results suggest that hepsin-directed therapy could be effective in prostate and breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4827. doi:1538-7445.AM2012-4827