Identification of TNO155, an Allosteric SHP2 Inhibitor for the Treatment of Cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy.

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.

Structure-Guided Design of EED Binders Allosterically Inhibiting the Epigenetic Polycomb Repressive Complex 2 (PRC2) Methyltransferase.

PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis.

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5' splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were "rescued" in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis.

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non-small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977-89. ©2016 AACR.

Modified Seed Growth of Iron Oxide Nanoparticles in Benzyl Alcohol - Optimization for Heating and Broad Stability in Biomedical Applications.

Iron oxide nanoparticles have received sustained interest for biomedical applications as synthetic approaches are continually developed for control of nanoparticle properties. However, many approaches focus solely on the material, rather than the complete optimization of synthesis and functionalization together to enhance translation into biological systems. Presented herein is a modified seed growth method designed for obtaining optimal nanoparticle properties and ease of surface functionalization for long term stability. With a one or two addition process, iron oxide nanoparticles were produced in crystallite sizes ranging from 5-15 nm using only benzyl alcohol and an iron precursor. In the functionalization process, concentration variations were required for stabilizing different nanoparticle sizes. Radio frequency induction heating experiments of various crystallite and hydrodynamic sizes verified that the heating efficiency greatly increased while approaching the 15 nm crystallite, and suggested an important role of the overall particle size on heating efficiency. Initial in vitro experiments with the functionalized nanoparticles showed success in providing hyperthermia-induced tumour cell killing without an increase in the temperature of the cell suspension medium. This demonstrates the potential for nanoparticle-based hyperthermia to provide a therapeutic effect while limiting normal tissue damage.

hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, which are regulated by RNA trans-factors associated with exon 3 of caspase-9 pre-mRNA (C9/E3). In this study, we identified hnRNP U as an RNA trans-factor associated with C9/E3. Down-regulation of hnRNP U led to a decrease in the caspase-9a/9b mRNA ratio, demonstrating a novel enhancing function. Importantly, hnRNP U bound specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interfered with hnRNP U binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Taken together, these findings show that hnRNP U competes with hnRNP L for binding to C9/E3 to enhance the inclusion of the four-exon cassette, and this splice-enhancing function is blocked by the AKT pathway via phosphorylation of hnRNP L.

Metallofullerene-nanoplatform-delivered interstitial brachytherapy improved survival in a murine model of glioblastoma multiforme.

Fullerenes are used across scientific disciplines because of their diverse properties gained by altering encapsulated or surface-bound components. In this study, the recently developed theranostic agent based on a radiolabeled functionalized metallofullerene ((177)Lu-DOTA-f-Gd(3)N@C(80)) was synthesized with high radiochemical yield and purity. The efficacy of this agent was demonstrated in two orthotopic xenograft brain tumor models of glioblastoma multiforme (GBM). A dose-dependent improvement in survival was also shown. The in vivo stability of the agent was verified through dual label measurements of biological elimination from the tumor. Overall, these results provide evidence that nanomaterial platforms can be used to deliver effective interstitial brachytherapy.

The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA.

Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 5'SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 µmol/L) or the pan-protein kinase C (PKC) inhibitor Gö6983 (25 µmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5'SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.

Biocatalytic synthesis and structure elucidation of cyclized metabolites of the deacetylase inhibitor panobinostat (LBH589).

Panobinostat (LBH589) is a novel pan-deacetylase inhibitor that is currently being evaluated in phase III clinical trials for treatment of Hodgkin's lymphoma and multiple myeloma. Under catalysis of recombinant human CYP3A4 and CYP2D6 coexpressed with human cytochrome P450 reductase in Escherichia coli JM109, five metabolites of panobinostat were produced via whole-cell biotransformation. The structures of the metabolites were elucidated with the spectroscopic methods mass spectrometry (MS) and NMR and revealed an oxidative cyclization of the ethyl-amino group to the methylindole moiety. The MS(2) spectrum of the cyclized metabolite showed a base peak, where the closed ring is reopened and that, taken as sole base for structure proposals, would have lead to wrong conclusions. The metabolites were substantially less potent deacetylase inhibitors than the parent compound.

Structure of human tankyrase 1 in complex with small-molecule inhibitors PJ34 and XAV939.

The crystal structures of tankyrase 1 (TNKS1) in complex with two small-molecule inhibitors, PJ34 and XAV939, both at 2.0 Å resolution, are reported. The structure of TNKS1 in complex with PJ34 reveals two molecules of PJ34 bound in the NAD(+) donor pocket. One molecule is in the nicotinamide portion of the pocket, as previously observed in other PARP structures, while the second molecule is bound in the adenosine portion of the pocket. Additionally, unlike the unliganded crystallization system, the TNKS1-PJ34 crystallization system has the NAD(+) donor site accessible to bulk solvent in the crystal, which allows displacement soaking. The TNKS1-PJ34 crystallization system was used to determine the structure of TNKS1 in complex with XAV939. These structures provide a basis for the start of a structure-based drug-design campaign for TNKS1.

[1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazoles: antagonists of the Wnt pathway that inhibit tankyrases 1 and 2 via novel adenosine pocket binding.

The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.

Metallofullerene-based nanoplatform for brain tumor brachytherapy and longitudinal imaging in a murine orthotopic xenograft model.

PURPOSE: To demonstrate in an orthotopic xenograft brain tumor model that a functionalized metallofullerene (f-Gd3N@C80) can enable longitudinal tumor imaging and, when radiolabeled with lutetium 177 (¹77Lu) and tetraazacyclododecane tetraacetic acid (DOTA) (¹77Lu-DOTA-f-Gd3N@C80), provide an anchor to deliver effective brachytherapy. MATERIALS AND METHODS: All experiments involving the use of mice were carried out in accordance with protocols approved by the institutional animal care and use committee. Human glioblastoma U87MG cells were implanted by using stereotactic procedures into the brains of 37 female athymic nude-Foxn1nu mice and allowed to develop into a tumor for 8 days. T1- and T2-weighted magnetic resonance (MR) imaging was performed in five mice. Biodistribution studies were performed in 12 mice at four time points over 7 days to evaluate gadolinium content. Survival studies involved 20 mice that received infusion of a nanoplatform by means of convection-enhanced delivery (CED) 8 days after tumor implantation. Mice in survival studies were divided into two groups: one comprised untreated mice that received f-Gd3N@CC80 alone and the other comprised mice treated with brachytherapy that received 1.11 MBq of ¹77Lu-DOTA-f-Gd3N@CC80. Survival data were evaluated by using Kaplan-Meier statistical methods. RESULTS: MR imaging showed extended tumor retention (25.6% ± 1.2 of the infused dose at 52 days, confirmed with biodistribution studies) of the f-Gd3N@CC80 nanoplatform, which enabled longitudinal imaging. Successful coupling of ¹77Lu to the f-Gd3N@CC80 surface was achieved by using a bifunctional macrocyclic chelator. The extended tumor retention allowed for effective brachytherapy, as indicated by extended survival time (> 2.5 times that of the untreated group) and histologic signs of radiation-induced tumor damage. CONCLUSION: The authors have developed a multimodal nanoplatform and have demonstrated longitudinal tumor imaging, prolonged intratumoral probe retention, biodistribution, and extended survival in an orthotopic xenograft brain tumor model.

Optimization of the in vitro cardiac safety of hydroxamate-based histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors have shown promise in treating various forms of cancer. However, many HDAC inhibitors from diverse structural classes have been associated with QT prolongation in humans. Inhibition of the human ether a-go-go related gene (hERG) channel has been associated with QT prolongation and fatal arrhythmias. To determine if the observed cardiac effects of HDAC inhibitors in humans is due to hERG blockade, a highly potent HDAC inhibitor devoid of hERG activity was required. Starting with dacinostat (LAQ824), a highly potent HDAC inhibitor, we explored the SAR to determine the pharmacophores required for HDAC and hERG inhibition. We disclose here the results of these efforts where a high degree of pharmacophore homology between these two targets was discovered. This similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be highly efficacious with weak affinity for the hERG and other ion channels.

Conjugation of functionalized gadolinium metallofullerenes with IL-13 peptides for targeting and imaging glial tumors.

BACKGROUND: Glioblastoma multiforme is the most common and most lethal primary brain tumor in humans, with median survival of approximately 1 year. Owing to the ability of glioma cells to aggressively infiltrate normal brain tissue and survive exposure to current adjuvant therapies, there is a great need for specific targeted nanoplatforms capable of delivering both therapeutic and imaging agents directly to invasive tumor cells. METHOD: Gadolinium-containing endohedral fullerenes, highly efficient contrast agents for MRI, were functionalized and conjugated with a tumor-specific peptide and assessed for their ability to bind to glioma cells in vitro. RESULTS: We report the successful conjugation of the carboxyl functionalized metallofullerene Gd(3)N@C(80)(OH)(-26)(CH(2)CH(2)COOH)(-16) to IL-13 peptides and the successful targeting ability towards brain tumor cells that overexpress the IL-13 receptor (IL-13Rα2). CONCLUSION: These studies demonstrate that IL-13 peptide-conjugated gadolinium metallofullerenes could serve as a platform to deliver imaging and therapeutic agents to tumor cells.

Encapsulation of a radiolabeled cluster inside a fullerene cage, (177)Lu(x)Lu((3-x))N@C(80): an interleukin-13-conjugated radiolabeled metallofullerene platform.

In this communication, we describe the successful encapsulation of (177)Lu into the endohedral metallofullerene (177)Lu(x)Lu(3-x)N@C(80) (x = 1-3) starting with (177)LuCl(3) in a modified quartz Kraschmer-Huffman electric generator. We demonstrate that the (177)Lu (beta-emitter) in this fullerene cage is not significantly released for a period of up to at least one-half-life (6.7 days). We also demonstrate that this agent can be conjugated with an interleukin-13 peptide that is designed to target an overexpressed receptor in glioblastoma multiforme tumors. This nanoparticle delivery platform provides flexibility for a wide range of radiotherapeutic and radiodiagnostic multimodal applications.

Reactive nature of dopamine as a surface functionalization agent in iron oxide nanoparticles.

Dopamine forms an initial structure coordinated to the surface of the iron oxide nanoparticle as a result of improved orbital overlap of the five-membered ring and a reduced steric environment of the iron complex. However, through transfer of electrons to the iron cations on the surface and rearrangement of the oxidized dopamine, a semiquinone is formed. Because of free protons in the system, oxygens on the surface are protonated, which allows for the Fe2+ to be released into the solution as a hydroxide. This released fragment of the nanoparticle will then eventually oxidize in air to a form of an iron(III) oxyhydroxide. All of the reported results demonstrate that the reactivity between Fe3+ and dopamine quickly facilitates the degradation of the nanoparticles. The energetic modeling studies substantiate our proposed decomposition mechanism and thus conclude that the use of dopamine as a robust anchor for iron oxide or iron oxide shell particles will not fulfill the need for stable ferrofluids in most biomedical applications.

Interfacial peptide inhibitors of HIV-1 integrase activity and dimerization.

Peptides derived from the interfacial region of dimeric HIV-1 integrase were evaluated as inhibitors of integrase's 3'-endonuclease activity. Three peptides were found to be moderately potent inhibitors with IC(50) values in the low micromolar range. The mode of inhibition was probed through protein crosslinking experiments. Active interfacial peptides were found to inhibit crosslinking of the dimeric form of integrase. Interfacial peptides that were poor inhibitors had no effect on integrase crosslinking.