2019 AD/PD: Discovering small-molecule modulators of autophagy for neurodegeneration

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Discovering Small-Molecule Modulators of Autophagy for Neurodegeneration

Summary

  • Autophagy is a major protein degradation pathway with proven roles in protecting neurons against accumulation of aggregation-prone proteins and obsolete mitochondria. Genetics of human-disease and mouse-knockout studies highlight the connection between autophagy dysfunction and neurodegeneration, and therefore finding a way to augment autophagic-flux seems a promising therapeutic strategy (Rubinsztein DC, Bento CF & Deretic V. Journal of Experimental Medicine 2015; Bento CF et al. Annual Review of Biochemistry 2016).
  • Fragment-based drug discovery (FBDD) has the potential to deliver potent, selective and CNS-penetrant small-molecules capable of inducing autophagy in the brain. For that purpose, the use of cell-based assays that accurately monitor the ability of small-molecules to modulate autophagy in a high-throughput- and unbiased-manner is instrumental.

2018 EORTC: A novel ERK1/2 inhibitor has potent activity in NRAS-mutant melanoma cancer models

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A novel ERK1/2 inhibitor has potent activity in NRAS-mutant melanoma cancer models

Summary

  • NRAS mutations occuring 15- 20% of melanoma cancer patients. Currently there is no approved molecularly targeted therapy for NRAS mutant melanoma, an indication which is associated with aggressive clinical outcome and a poor prognosis.
  • The NRAS mutation leads to constitutive activation of the MAPK pathway. AsERK1/2(ERK) is the primary downstream effect or of the MAPK pathway, its direct inhibition may provide an attractive therapeutic approach for the treatment of NRAS-mutant melanoma.
  • As previously described, using fragment-based drug discovery we have identified a novel and selective inhibitor of ERK which inhibits in vitro ERK catalytic activity as well as ERK phosphorylation 1.
  • Here, we demonstrate the in vitro and in vivo activity of a novel, highly potent, selective ERK inhibitor in models of NRAS-mutant melanoma.

2017 EORTC: Characterisation of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2

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Characterisation of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2

Summary

  • The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF and RAS, and multiple studies have demonstrated that MAPK pathway inhibition suppresses the growth of such cancer cells.
  • MAPK inhibition has been clinically validated by BRAF and MEK inhibitors, which are approved for the treatment of BRAFV600E-mutant melanoma and NSCLC. However, response to such agents is often short-lived due to the onset of resistance mechanisms that result in re-activation of ERK1/2 (ERK) signalling1,2.
  • RAF and MEK inhibitors have also been clinically tested in other cancers, including BRAF-mutant colorectal (CRC) and KRAS-mutantNon-small cell lung cancer (NSCLC), where they had limited clinical activity3,4.
  • As ERK is the primary downstream effector of the MAPK pathway, it is hypothesized that ERK inhibitors may prove to be less susceptible to oncogenic bypass than RAF and MEK inhibitors and therefore have the potential to overcome the limitations of RAF and MEK inhibitors.
  • Using fragment-based drug discovery we have developed a novel, potent and selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nMIC50 value and has strong anti-proliferative effects in a wide range of MAPK-activated cell lines.
  • In addition to inhibiting ERK catalytic activity, the compound also inhibits the phosphorylation of ERK by MEK and confers a decrease in cellular pERKlevels in both BRAF-mutant and KRAS-mutant cell lines (in vitro and in in vivo pharmacodynamic[PD] studies).
  • Once daily oral dosing of the lead compound (50 mg/kg) conferred significant anti-tumoractivity in a range of in vivo models.
  • These data support the further optimisation of this series of compounds for clinical development.

2020 EHA: Characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, in mouse models of T cell lymphoma

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Characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, in mouse models of T cell lymphoma

Abstract:

Background:  ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumor and lymphomas (NCT02503423). IAP antagonists have been reported to exhibit broad immuno-modulatory effects on both the innate and adaptive immune systems .

Aims:  We have investigated the profile of ASTX660 in preclinical T cell lymphoma models and evaluated ASTX660’s ability to enhance immune mediated killing of T cell lymphoma cells, both in vitro and in vivo.

Methods:  ASTX660 was tested in a panel of human and mouse tumor cell lines, assessing apoptosis, necroptosis and immunogenic cell death (ICD). ASTX660 was tested in vitro alone or with recombinant death receptor ligands (TNFa, FASL or TRAIL) and with or without caspase-8/RIPK inhibitors to demonstrate mechanism of action. Target engagement along with induction of apoptosis, necroptosis and ICD markers were analysed by Western blotting, and flow cytometry. Murine tumor models in immunocompetent and immunocompromised mice were utilised to evaluate the efficacy of ASTX660 in the presence or absence of an effective immune response. The Nanostring IO360 panel was used to assess immune cell recruitment.

Results:  ASTX660 antagonised IAPs in cell lines, as indicated by a decrease in cIAP1 protein levels and disruption of the XIAP:SMAC protein complex. In murine T cell lymphoma cell lines (BW5147, EL4 and L5178Y), ASTX660 treatment was associated with an increase in apoptosis or necroptosis and ICD biomarkers. In immunocompetent mice, administration of ASTX660 delivered a complete regression of BW5147 tumor growth, which was not seen in mice deficient in T and B cells. These mice remained refractory to subsequent rechallenge after initial complete regression. Biomarker evaluation from this model indicated a potent immunogenic/necroptotic response after ASTX660 dosing and upregulation of immune effector cells.

References:
1. Michie J. et al., The Immuno-Modulatory Effects of Inhibitor of Apoptosis Proteins Antagonists, Cells, 2020, 9(1), 207.
2. A. Hollebecque et al., AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2019.

 

2020 EHA: ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine

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ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine

Abstract:

Background: The tumour suppressor p53 is activated in response to various stress signals to induce transcriptional changes leading to cellular responses such as cell cycle arrest and apoptosis. Activity of p53 is tightly regulated by the E3 ubiquitin ligase MDM2, which inhibits p53 function by, for example, targeting it for proteasomal degradation. Targeting the MDM2-p53 interaction to restore p53 function, is therefore, a promising strategy for cancer therapy and a number of these compounds are in clinical development including ASTX295 (NCT03975387). ASTX295 is a novel, orally bioavailable MDM2 antagonist developed through structure-based drug design that has demonstrated potent activity in a range of p53 wild-type pre-clinical models.

Aims: We investigated the therapeutic potential of ASTX295 alone and in combination with decitabine, a DNA- hypomethylating agent, in AML.

Methods: Primary blasts were isolated from AML patient samples using a combination of antibodies against CD34, CD33, CD45 and CD117. A panel of AML cell lines and primary AML blasts were treated with decitabine and ASTX295 at a range of concentrations, alone and in combination. After treatment, viability was assessed by Alamar blue assay or induction of apoptosis by flow cytometry using a fluorescent caspase substrate or Annexin V.

Effects of drug combinations were analysed using the Combenefit software based on different mathematical models (Loewe, bliss & HSA). Target engagement was confirmed by western blotting.

Results: When tested in a panel of p53 wild-type AML cell lines, ASTX295 exerted a strong anti-proliferative effect in which GI50 <30 nM was observed in 9 out of 11 cell lines. Additionally, p53 activation by ASTX295 triggered apoptosis in both AML cell lines, and primary AML blasts isolated from patients.

Activity of ASTX295 was further enhanced by combining with decitabine. Treatment of AML cell lines with ASTX295 and decitabine showed an increase in growth inhibitory effect and apoptosis compared to respective single agent treatments. This combinatory effect, as assessed by Combenefit, was also observed in primary AML blasts in which 7 of 12 samples tested demonstrated increased apoptosis at or above 300 nM ASTX295 and 100 nM decitabine. Target engagement of ASTX295 and decitabine was confirmed by upregulation of p53 transcriptional targets and decreased DNMT-1 expression.

Summary/Conclusion: Our findings demonstrate that the combination of ASTX295 with decitabine exhibits potent activity against p53 wild-type AML cells, and thus merits further investigation.

2018 AACR: Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction

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Summary
In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development.

An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity.  This approach led to potent compounds with EC50 <1 nM against MDM2 in cell-free ELISA assays and EC50 <30 nM for p53 induction in SJSA-1 osteosarcoma cells. Further analyses of the compounds demonstrated an increase in the levels of p53 and p53 transcriptional targets as a result of inhibiting the MDM2-p53 interaction. Using three pairs of isogenic cell lines, the compounds were shown to be specific for cell lines with wild-type p53. Key compounds were also characterized in pharmacokinetic and pharmacodynamic studies in mice bearing the SJSA-1 tumor xenograft where they displayed strong induction of p53, 3 hours post oral administration, together with an increase in the expression of p53 target genes p21 and MDM2. These potent MDM2-p53 inhibitors have also shown significant in vivo efficacy in the SJSA-1 xenograft model at well tolerated oral doses.  Thus, promising lead compounds were identified, meriting further optimization of the series.

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Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction

Characterisation of fragments binding to the translation initiation factor eIF4E

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Summary

  • Eukaryotic translation initiation factor 4E (eIF-4E) is a key component of the m7G-cap-binding protein complex eIF-4F and is required for capdependent
    translation initiation. Activity of the eIF4F complex is tightly controlled by both the PI3K/Akt/mTOR and Raf/Mek/ERK pathways,
    via mTOR phosphorylation of the eIF4E sequestering proteins 4E-BP1-3 and phosphorylation of eIF4E by MNK1/2, downstream of ERK. EIF4E
    is therefore a key node downstream of pathways that are frequently dysregulated in cancer.
  • Formation of the eIF4F complex leads to translation of ‘weak’ mRNAs, encoding key cell growth and survival proteins such as cyclin D1,
    c-MYC and Mcl1, supporting cancer cell proliferation, and has been associated with resistance to MAPK and PI3K inhibitors1,2. Identification
    of an inhibitor of eIF4E would therefore be of therapeutic value.
  •  The Astex fragment screening platform was used to identify fragment hits binding to an unprecedented binding site on eIF4E. These weak
    hits were optimised using structure guided design into functional effects on cap dependent translation by inhibiting the formation of eIF4F translation initiation complex.

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Characterisation of fragments binding to the translation initiation factor eIF4E

Fragment Based Drug Discovery: An Organic Synthesis Perspective

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Summary

  •  Fragment hits bind with high energy interactions, so have high ligand efficiency (LE).
  • Small libraries of fragments can sample chemical space more widely There are estimated to be ~ 1050 compounds < 500 MW, cf.
    ~106 fragments < 250 MW.
  • Potencies in mM rather than μM range require sensitive biophysical techniques to detect interactions, e.g. X-Ray, NMR, SPR, ITC.
  • Millimolar fragments can be converted to nanomolar leads with the  support of structure based drug design.

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Fragment Based Drug Discovery: An Organic Synthesis Perspective

TAT: Anti-Angiogenic Activity of Fragment-Derived Inhibitors of METAP2

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Summary

Methionine amino peptidase (MetAP) 2 is the target of the anti- angiogenic natural product fumagillin and so is believed to play a role in angiogenesis. MetAPs are metalloenzymes which cleave the N- terminal methionine from newly formed polypeptides. This allows essential post-translational modifications, such as myristoylation and acetylation, to take place thus generating fully functional proteins.
Analogues of fumagillin have shown activity in several different disease models, where angiogenesis may be relevant, including
oncology. Semi-synthetic analogues of fumagillin, such as TNP-470, have shown evidence of antitumor activity in the clinic but poor pharmacokinetic properties and neurotoxic effects have limited their development. Nevertheless MetAP2 remains a promising oncology
target and inhibitors with improved properties should have potential as anti-angiogenic agents.
We have screened MetAP2 using our fragment-based screening approach (Pyramid™) and identified multiple low molecular weight fragments, which bind at the active site of MetAP2 in diverse ways. Three of these were optimised to novel hit series using structure- based drug design and their anti-angiogenic properties are described here.

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TAT: Anti-Angiogenic Activity of Fragment-Derived Inhibitors of METAP2