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.

Randomized window of opportunity trial evaluating high-dose vitamin D in breast cancer patients.

PURPOSE: Epidemiologic and preclinical data suggest a potential role for vitamin D in breast cancer treatment and prevention. However, results of prospective randomized trials are inconsistent. The objective of this study was to assess the effects of high-dose cholecalciferol (vitamin D3) on breast tumour proliferation and apoptosis. METHODS: We conducted a prospective, randomized, phase 2, double-blinded pre-surgical window of opportunity trial. Newly diagnosed breast cancer patients were randomized to receive 40,000 IU of vitamin D3 per day or placebo for 2 to 6 weeks prior to breast surgery. The primary outcome was the relative change in proliferation (Ki67) and apoptosis (cleaved caspase 3 apoptotic assay [CC3]) in primary breast cancer cells pre and post treatment. RESULTS: Of 83 patients randomized, 80 completed the study (43 (53.8%) vitamin D and 37 (46.3%) placebo). Mean duration of drug intake was 19 days (range 9-28 days). There were no significant differences between the control arm and the vitamin D arm in percent changes of either Ki67 index (1.6% vs. 16.7%, p = 0.25) or CC3 (- 55.9% vs. - 45.9%, p = 0.28). Serum 25-hydroxyvitamin D (25-OHD) levels were 3 times higher in the vitamin D arm (62 nmol/L vs. 246 nmol/L, p < 0.001). Adverse effects were minimal and all classified as grade 1. CONCLUSIONS: Despite significantly higher levels of serum 25-OHD in the vitamin D-treated group, this was not associated with any significant effects on tumour proliferation or apoptosis. These findings are consistent with the lack of benefit observed in prospective prevention trials. TRIAL REGISTRY: Trial registration clinicaltrials.gov NCT01948128.

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).

Cryptosphaerolide, a cytotoxic Mcl-1 inhibitor from a marine-derived ascomycete related to the genus Cryptosphaeria.

Examination of the saline fermentation products from the marine-derived ascomycete fungal strain CNL-523 (Cryptosphaeria sp.) resulted in the isolation of cryptosphaerolide (1). The new compound is an ester-substituted sesquiterpenoid related to the eremophilane class. The structure of the new compound was assigned by spectroscopic and chemical methods. Cryptosphaerolide was found to be an inhibitor of the protein Mcl-1, a cancer drug target involved in apoptosis. It also showed significant cytotoxicity against an HCT-116 human colon carcinoma cell line, indicating that the compound may be of value in exploring the Mcl-1 pathway as a target for cancer chemotherapy.

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.

Genetic predictors of antipsychotic response to lurasidone identified in a genome wide association study and by schizophrenia risk genes.

Biomarkers which predict response to atypical antipsychotic drugs (AAPDs) increases their benefit/risk ratio. We sought to identify common variants in genes which predict response to lurasidone, an AAPD, by associating genome-wide association study (GWAS) data and changes (Δ) in Positive And Negative Syndrome Scale (PANSS) scores from two 6-week randomized, placebo-controlled trials of lurasidone in schizophrenia (SCZ) patients. We also included SCZ risk SNPs identified by the Psychiatric Genomics Consortium using a polygenic risk analysis. The top genomic loci, with uncorrected p<10-4, include: 1) synaptic adhesion (PTPRD, LRRC4C, NRXN1, ILIRAPL1, SLITRK1) and scaffolding (MAGI1, MAGI2, NBEA) genes, both essential for synaptic function; 2) other synaptic plasticity-related genes (NRG1/3 and KALRN); 3) the neuron-specific RNA splicing regulator, RBFOX1; and 4) ion channel genes, e.g. KCNA10, KCNAB1, KCNK9 and CACNA2D3). Some genes predicted response for patients with both European and African Ancestries. We replicated some SNPs reported to predict response to other atypical APDs in other GWAS. Although none of the biomarkers reached genome-wide significance, many of the genes and associated pathways have previously been linked to SCZ. Two polygenic modeling approaches, GCTA-GREML and PLINK-Polygenic Risk Score, demonstrated that some risk genes related to neurodevelopment, synaptic biology, immune response, and histones, also contributed to prediction of response. The top hits predicting response to lurasidone did not predict improvement with placebo. This is the first evidence from clinical trials that SCZ risk SNPs are related to clinical response to an AAPD. These results need to be replicated in an independent sample.

Dual Allosteric Inhibition of SHP2 Phosphatase.

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.

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.

Estrogen receptor beta isoforms exhibit differences in ligand-activated transcriptional activity in an estrogen response element sequence-dependent manner.

Estrogen receptor beta (ERbeta) has been reported to have lower estradiol (E2)-induced transcriptional activity than human (h)ERalpha from estrogen response element (ERE)-driven reporters in transiently transfected cells. Conflicting data for activities of full-length and short hERbeta [hERbeta1, 530 amino acids (aa); and hERbeta1s, 477aa] have been reported. To test the hypothesis that hERbeta1 has higher transcriptional activity than hERbeta1s, we compared E2, 2,3-bis(4-hydroxyphenyl)propionitrile (a selective ERbeta agonist), and resveratrol-induced transcription by hERbeta1, hERbeta1s, and rat (r) ERbeta with hERalpha on different EREs in transiently transfected CHO-K1 and HEC-1A cells. Our results demonstrate for the first time that hERbeta1 has similar E2-induced activity to hERalpha and greater activity than rERbeta or hERbeta1s on a consensus palindromic ERE, either as a single or double copy; a minimal ERE; and the nonpalindromic pS2 ERE. 2,3-Bis(4-hydroxyphenyl)propionitrile showed greater efficacy with hERbeta1 and hERbeta1s than for rERbeta or hERalpha. We found that transcriptional differences between the ERbeta isoforms and ERalpha depend on the ERE sequence, confirming that the DNA sequence bound by ER is an allosteric effector of ER action. For the minimal 13-bp ERE and the pS2 ERE, the increase in transcriptional activity with hERbeta1 correlated with increased binding affinity. Coactivators steroid receptor coactivator-1 and cAMP response element binding protein-binding protein synergistically activated hERalpha and ERbeta transcription and showed reduced efficacy with rERbeta and hERbeta1s, suggesting a role for the N terminus of ERbeta1 in coactivator interaction. Collectively, these data indicate that the cellular expression of ERbeta isoforms may differentially impact ERE-regulated target gene expression in a ligand-dependent manner.

Glucagon-like peptide 1 receptor (GLP1R) haplotypes correlate with altered response to multiple antipsychotics in the CATIE trial.

Glucagon-like peptide 1 receptor (GLP1R) signaling has been shown to have antipsychotic properties in animal models and to impact glucose-dependent insulin release, satiety, memory, and learning in man. Previous work has shown that two coding mutations (rs6923761 and rs1042044) are associated with altered insulin release and cortisol levels. We identified four frequently occurring haplotypes in Caucasians, haplotype 1 through haplotype 4, spanning exons 4-7 and containing the two coding variants. We analyzed response to antipsychotics, defined as predicted change in PANSS-Total (dPANSS) at 18 months, in Caucasian subjects from the Clinical Antipsychotic Trial of Intervention Effectiveness treated with olanzapine (n=139), perphenazine (n=78), quetiapine (n=14), risperidone (n=143), and ziprasidone (n=90). Haplotype trend regression analysis revealed significant associations with dPANSS for olanzapine (best p=0.002), perphenazine (best p=0.01), quetiapine (best p=0.008), risperidone (best p=0.02), and ziprasidone (best p=0.007). We also evaluated genetic models for the two most common haplotypes. Haplotype 1 (uniquely including the rs1042044 [Leu(260)] allele) was associated with better response to olanzapine (p=0.002), and risperidone (p=0.006), and worse response to perphenazine (p=.03), and ziprasidone (p=0.003), with a recessive genetic model providing the best fit. Haplotype 2 (uniquely including the rs6923761 [Ser(168)] allele) was associated with better response to perphenazine (p=0.001) and worse response to olanzapine (p=.02), with a dominant genetic model providing the best fit. However, GLP1R haplotypes were not associated with antipsychotic-induced weight gain. These results link functional genetic variants in GLP1R to antipsychotic response.

Replication of SULT4A1-1 as a pharmacogenetic marker of olanzapine response and evidence of lower weight gain in the high response group.

AIM: Antipsychotic efficacy biomarkers have the potential to improve outcomes in psychotic patients. This study examined the effect of SULT4A1-1 haplotype status (rs2285162 [A]-rs2285167 [G]) on olanzapine response. PATIENTS & METHODS: We evaluated 87 olanzapine treated subjects from Phases 1, 1B and 2 of the CATIE trial for the impact of SULT4A1-1 status on change in Positive and Negative Syndrome Scale (PANSS) total score using two models of response. We also examined weight change. RESULTS: SULT4A1-1-positive status correlated with superior olanzapine response in Phase 1 (p = 0.004 for model 1 and p = 0.001 for model 2) and Phases 1B/2 (p = 0.05 for model 1 and p = 0.007 for model 2). SULT4A1-1-positive subjects gained significantly less weight per month on olanzapine, 0.15 lbs, than did SULT4A1-1-negative subjects, 2.27 lbs (p = 0.04). CONCLUSION: This study provides a second replication of superior olanzapine response in SULT4A1-1-positive subjects compared with SULT4A1-1-negative subjects. SULT4A1-1-positive subjects treated with olanzapine also gained less weight than SULT4A1-1-negative subjects.

Genotypic variation in the SV2C gene impacts response to atypical antipsychotics the CATIE study.

Pharmacogenetic (PGx) predictors of response would improve outcomes in antipsychotic treatment. Based on both biological rationale and prior evidence of an impact on Parkinson's disease, we conducted an association study for 106 SNPs in the synaptic vesicle protein 2C (SV2C) gene using genetic and treatment response data from the Clinical Trial of Antipsychotic Intervention Effectiveness (CATIE). We examined response to the atypical antipsychotics for Caucasian subjects in the blinded phases, Phases 1A, 1B, and 2, of CATIE with sample sizes as follows: olanzapine (N=134), quetiapine (N=124), risperidone (N=134), and ziprasidone (N=74). Response was defined as change in the Positive and Negative Syndrome Scale (PANSS) score using a mixed model repeat measures approach. Subjects homozygous for the T allele of rs11960832 displayed significantly worse response to olanzapine treatment, the only finding with study-wide significance (p=2.94×10(-5); false discovery rate=2.18×10(-2)). These subjects also displayed worse response to quetiapine with nominal significance (p=4.56×10(-2)). While no other SNP achieved study-wide significance, one SNP (rs10214163) influencing Parkinson's disease displayed nominally significant association with olanzapine and quetiapine response, while the second such SNP (rs30196) showed a statistical trend toward correlating with olanzapine and quetiapine response. Furthermore, both coding SNPs examined (rs31244 and rs2270927) displayed nominally significant correlations with treatment response: one for olanzapine (rs227092), and one for quetiapine (rs31244). The fact that multiple SNPs in SV2C may impact response to atypical antipsychotics suggests that further evaluation of SNPs in this gene as PGx predictors of antipsychotic response is warranted.

Sulfotransferase 4A1 Haplotype 1 (SULT4A1-1) Is Associated With Decreased Hospitalization Events in Antipsychotic-Treated Patients With Schizophrenia.

OBJECTIVE: To evaluate a common genetic variant, sulfotransferase 4A1 haplotype 1 (SULT4A1-1), as a predictor of hospitalization events due to the exacerbation of schizophrenia for patients treated with antipsychotic medications. Haplotypes were determined using single nucleotide polymorphism data. METHOD: The study included 417 white subjects from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study with a DSM-IV diagnosis of schizophrenia. Patients were assigned to 1 of 4 atypical antipsychotics (olanzapine, quetiapine, risperidone, or ziprasidone) or to the first-generation antipsychotic perphenazine. Kaplan-Meier survival analysis and Cox proportional hazards regression models were used to measure if haplotype status impacted hospitalization events for these 5 treatments. Haplotype status was evaluated for its relationship to hospitalization events regardless of treatment and for the individual treatments, with or without previous exacerbation. Data for the CATIE study were collected from January 2001 to December 2004. The current post hoc analysis was performed between May 2011 and August 2011. RESULTS: In phase 1 of the trial, considering only the first hospitalization events, the haplotype had a significant impact on hospitalization events, with a hazard ratio for SULT4A1-1(-) versus SULT4A1-1(+) of 2.54 (P = .048). When prior exacerbation was included in the model for phase 1, the hazard ratio was 2.34 (P = .072) considering only the first hospitalization event and 2.71 (P = .039) considering all hospitalization events in the phase. When data for all phases were evaluated, SULT4A1-1(-) status was associated with increased hospitalization risk for subjects treated with olanzapine, with a hazard ratio of 8.26 (P = .041), and possibly for subjects treated with quetiapine, with a hazard ratio of 6.80 (P = .070). CONCLUSIONS: The SULT4A1-1 haplotype may be an important predictor of risk of hospitalization. SULT4A1-1(+) status was significantly associated with decreased risk of hospitalization when the subjects were treated with olanzapine.

Evidence for a SULT4A1 haplotype correlating with baseline psychopathology and atypical antipsychotic response.

AIM: This study evaluated the impact of SULT4A1 gene variation on psychopathology and antipsychotic drug response in Caucasian subjects from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study and a replication sample. PATIENTS & METHODS: SULT4A1 haplotypes were determined using SNP data. The relationship to baseline psychopathology was evaluated using linear regression of Positive and Negative Syndrome Scale (PANSS) total score. Drug response was evaluated using Mixed Model Repeat Measures (MMRM) for change in PANSS. RESULTS: For the CATIE sample, patients carrying a haplotype designated SULT4A1-1(+) displayed higher baseline PANSS (p = 0.03) and, when treated with olanzapine, demonstrated a significant interaction with time (p = 0.009) in the MMRM. SULT4A1-1(+) patients treated with olanzapine displayed improved response compared with SULT4A1-1(-) patients treated with olanzapine (p = 0.008) or to SULT4A1-1(+) patients treated with risperidone (p = 0.006). In the replication sample, SULT4A1-1(+) patients treated with olanzapine demonstrated greater improvement than SULT4A1-1(-) patients treated with olanzapine (p = 0.05) or than SULT4A1-1(+) patients treated with risperidone (p = 0.05). CONCLUSION: If validated, determination of SULT4A1-1 haplotype status might be useful for identifying patients who show an enhanced response to long-term olanzapine treatment. Original submitted 6 October 2010; Revision submitted 9 December 2010.

AAL881, a novel small molecule inhibitor of RAF and vascular endothelial growth factor receptor activities, blocks the growth of malignant glioma.

Malignant gliomas are highly proliferative and angiogenic cancers resistant to conventional therapies. Although RAS and RAF mutations are uncommon in gliomas, RAS activity is increased in gliomas. Additionally, vascular endothelial growth factor and its cognate receptors are highly expressed in gliomas. We now report that AAL881, a novel low-molecular weight inhibitor of the kinase activities associated with B-RAF, C-RAF (RAF-1), and VEGF receptor-2 (VEGFR2), showed activity against glioma cell lines and xenografts. In culture, AAL881 inhibited the downstream effectors of RAF in a concentration-dependent manner, with inhibition of proliferation associated with a G(1) cell cycle arrest, induction of apoptosis, and decreased colony formation. AAL881 decreased the proliferation of bovine aortic endothelial cells as well as the tumor cell secretion of vascular endothelial growth factor and inhibited the invasion of glioma cells through an artificial extracellular matrix. Orally administered AAL881 was well tolerated with minimal weight loss in non-tumor-bearing mice. Established s.c. human malignant glioma xenografts grown in immunocompromised mice treated with a 10-day course of oral AAL881 exhibited growth delays relative to control tumors, frequently resulting in long-term complete regressions. AAL881 treatment extended the survival of immunocompromised mice bearing orthotopic glioma xenografts compared with placebo controls. The intraparenchymal portions of orthotopic AAL881-treated tumors underwent widespread necrosis consistent with vascular disruption compared with the subarachnoid elements. These effects are distinct from our prior experience with VEGFR2 inhibitors, suggesting that targeting RAF itself or in combination with VEGFR2 induces profound tumor responses in gliomas and may serve as a novel therapeutic approach in patients with malignant gliomas.