Correction: Discovery and clinical introduction of first-in-class imipridone ONC201.

[This corrects the article DOI: 10.18632/oncotarget.11814.].

HLA-DR15-specific inhibition attenuates autoreactivity to the Goodpasture antigen.

Goodpasture's disease manifests as rapidly progressive glomerulonephritis. Current immunosuppressive treatments do not specifically target the pathological immune response and have significant side effects. Like most autoimmune diseases, the strongest genetic association is with the HLA alleles. Inheritance of HLA-DR15 confers susceptibility, and structure-function studies have shown that HLA-DR15 plays a causative role in activating autoreactive pro-inflammatory T cells. Thus, specific inhibition of HLA-DR15 would provide a targeted therapeutic approach. We hypothesised that PV-267, an HLA-DR15-specific inhibitor, would effectively block HLA-DR15 presentation of the dominant epitope, attenuate the activation of autoreactive T cells, and limit disease. Using humanised HLA-DR15 transgenic mice, α3135-145-specific, pro-inflammatory T cell recall responses were measured using IFN-γ and IL-17A ELISPOTs and by proliferation assay. To determine if PV-267 could limit disease, experimental autoimmune anti-GBM glomerulonephritis was induced in HLA-DR15 transgenic mice (on an Fcgr2b-/- background), and functional and histological disease endpoints were measured. PV-267 effectively inhibited α3135-145-specific immune responses and disease development. Mice treated prior to immunization with α3135-145 had reduced α3135-145-specific recall responses, and limited disease by albuminuria, histological glomerular injury, IgG deposition, and inflammatory cell infiltrates. PV-267 treatment commencing after the onset of active anti-α3(IV)NC1 autoimmunity attenuated functional and histological renal injury. When treatment was administered after disease was established, PV-267 limited the severity of histological injury. In conclusion, HLA-DR15 inhibition attenuates α3(IV)NC1-specific pro-inflammatory responses and could be used as an adjunct therapy for anti-GBM disease.

Preclinical evaluation of the imipridone family, analogs of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212.

Anti-cancer small molecule ONC201 upregulates the integrated stress response (ISR) and acts as a dual inactivator of Akt/ERK, leading to TRAIL gene activation. ONC201 is under investigation in multiple clinical trials to treat patients with cancer. Given the unique imipridone core chemical structure of ONC201, we synthesized a series of analogs to identify additional compounds with distinct therapeutic properties. Several imipridones with a broad range of in vitro potencies were identified in an exploration of chemical derivatives. Based on in vitro potency in human cancer cell lines and lack of toxicity to normal human fibroblasts, imipridones ONC206 and ONC212 were prioritized for further study. Both analogs inhibited colony formation, and induced apoptosis and downstream signaling that involves the integrated stress response and Akt/ERK, similar to ONC201. Compared to ONC201, ONC206 demonstrated improved inhibition of cell migration while ONC212 exhibited rapid kinetics of activity. ONC212 was further tested in >1000 human cancer cell lines in vitro and evaluated for safety and anti-tumor efficacy in vivo. ONC212 exhibited broad-spectrum efficacy at nanomolar concentrations across solid tumors and hematological malignancies. Skin cancer emerged as a tumor type with improved efficacy relative to ONC201. Orally administered ONC212 displayed potent anti-tumor effects in vivo, a broad therapeutic window and a favorable PK profile. ONC212 was efficacious in vivo in BRAF V600E melanoma models that are less sensitive to ONC201. Based on these findings, ONC212 warrants further development as a drug candidate. It is clear that therapeutic utility extends beyond ONC201 to include additional imipridones.

Discovery and clinical introduction of first-in-class imipridone ONC201.

ONC201 is the founding member of a novel class of anti-cancer compounds called imipridones that is currently in Phase II clinical trials in multiple advanced cancers. Since the discovery of ONC201 as a p53-independent inducer of TRAIL gene transcription, preclinical studies have determined that ONC201 has anti-proliferative and pro-apoptotic effects against a broad range of tumor cells but not normal cells. The mechanism of action of ONC201 involves engagement of PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt/ERK inactivation, and consequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animals models, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors confirmed that ONC201 is exceptionally well-tolerated and established the recommended phase II dose of 625 mg administered orally every three weeks defined by drug exposure comparable to efficacious levels in preclinical models. Clinical trials are evaluating the single agent efficacy of ONC201 in multiple solid tumors and hematological malignancies and exploring alternative dosing regimens. In addition, chemical analogs that have shown promise in other oncology indications are in pre-clinical development. In summary, the imipridone family that comprises ONC201 and its chemical analogs represent a new class of anti-cancer therapy with a unique mechanism of action being translated in ongoing clinical trials.

The angular structure of ONC201, a TRAIL pathway-inducing compound, determines its potent anti-cancer activity.

We previously identified TRAIL-inducing compound 10 (TIC10), also known as NSC350625 or ONC201, from a NCI chemical library screen as a small molecule that has potent anti-tumor efficacy and a benign safety profile in preclinical cancer models. The chemical structure that was originally published by Stahle, et. al. in the patent literature was described as an imidazo[1,2-a]pyrido[4,3-d]pyrimidine derivative. The NCI and others generally accepted this as the correct structure, which was consistent with the mass spectrometry analysis outlined in the publication by Allen et. al. that first reported the molecule's anticancer properties. A recent publication demonstrated that the chemical structure of ONC201 material from the NCI is an angular [3,4-e] isomer of the originally disclosed, linear [4,3-d] structure. Here we confirm by NMR and X-ray structural analysis of the dihydrochloride salt form that the ONC201 material produced by Oncoceutics is the angular [3,4-e] structure and not the linear structure originally depicted in the patent literature and by the NCI. Similarly, in accordance with our biological evaluation, the previously disclosed anti-cancer activity is associated with the angular structure and not the linear isomer. Together these studies confirm that ONC201, produced by Oncoceutics or obtained from the NCI, possesses an angular [3,4-e] structure that represents the highly active anti-cancer compound utilized in prior preclinical studies and now entering clinical trials in advanced cancers.

Small molecule inhibitor of antigen binding and presentation by HLA-DR2b as a therapeutic strategy for the treatment of multiple sclerosis.

The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.

Inhibition of amyloid beta-peptide production by blockage of beta-secretase cleavage site of amyloid precursor protein.

Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.

Designing isoform-specific peptide disruptors of protein kinase A localization.

A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.

Oxazole- and imidazole-based Ser-Leu dipeptide mimetics in potent inhibitors of antigen presentation by MHC class II DR molecules.

Imidazole and oxazole derivatives 1 to 4 were designed and prepared as dipeptide mimetics to replace the Ser-Leu dipeptide sequence of Ro-25-9980 (Ac-(Cha)-RAMA-S-L-NH2), a peptidic inhibitor of antigen binding to major histocompatibility complex (MHC) class II DR molecules linked to rheumatoid arthritis (RA). The most potent analog in binding assays (IC50 = 30 nM in DRB1*0401 binding; 1.6 times as potent as Ro 25-9980) was 16, Ac-(Cha)RAMA-(S)S-psi(oxazole)-L-NH2. The SAR of peptide hybrids 10 to 24, prepared by incorporating the dipeptide mimetics 1 to 4 is discussed. Of these hybrids, 23 and 24, analogs that incorporated the imidazole and oxazole mimetics as well as optimized variants at positions 3 to 5, were found to have 70 to 80 nM binding affinity comparable to the parent peptide in DRB 1*0401 binding and were also active in DRB1*0101 binding, while being resistant to proteolysis by cathepsin B. Both of these compounds showed inhibitory activity in an antigen-stimulated T-cell proliferation assay, indicating their potential to suppress autoimmune responses and as leads for therapeutic agents to treat RA.