File includes genomic analysis, cell cycle and cell proliferation analysis.

human Breast tumor Notch gene coverage annotations.

Table S1: Analysis of Notch gene rearrangements in triple negative breast cancer subtypes. Table S2: Genes whose expression correlates with Notch gene-rearrangement status in cancer cell lines. Table S3: Genes whose expression correlates with Notch gene-rearrangement in triple negative breast cancer. Table S4. Novel mutations identified in the NRR and PEST domains of NOTCH1 in solid tumors. Table S5: Notch NRR and PEST domain mutation frequency. Table S6: NOTCH1 mutational status, N1-ICD levels, MYC expression in Adenoid Cystic Carcinoma models. Table S7: Primary antibodies. Table S8: RT-PCR Primers.

S1: NOTCH gene rearrangement in breast cancer cell lines and human breast tumors. S2: NOTCH gene rearrangement and sensitivity to MRK-003. S3: HES4 expression is associated with NOTCH gene-rearrangement or activating mutation in NOTCH. S4: HES4 expression is a poor prognostic marker in TNBC. S5: Proliferation inhibition by combination therapy with MRK-003 and ERK inhibitor (SCH772984). S6: Effect of MRK-003 treatment on CD44+/CD24 high stem-like cell population. S7: N1ICD level correlates with mutation status and is predictive of MRK-003 response in xenograft models.

Human breast tumor GE Notch Pathway.

The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.

The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 μg mL-1 against fluoroquinolone-resistant Staphylococcus aureus. Although in vitro hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.

Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the discovery of REDX07965, which has an MIC90 of 0.5 μg mL-1 against Staphylococcus aureus, favourable in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity profile. The results herein validate a rational design approach to address the urgent unmet medical need for novel antibiotics.

According to the World Health Organization (WHO), approximately 1.7 million deaths per year are caused by tuberculosis infections. Furthermore, it has been predicted that, by 2050, antibacterial resistance will be the cause of approximately 10 million deaths annually if the issue is not tackled. As a result, novel approaches to treating broad-spectrum bacterial infections are of vital importance. During the course of our wider efforts to discover unique methods of targeting multidrug-resistant (MDR) pathogens, we identified a novel series of amide-linked pyrimido[4,5-b]indol-8-amine inhibitors of bacterial type II topoisomerases. Compounds from the series were highly potent against gram-positive bacteria and mycobacteria, with excellent potency being retained against a panel of relevant Mycobacterium tuberculosis drug-resistant clinical isolates.

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.

Abstract Harnessing the immune system via immune checkpoint blockade (e.g. anti PD-1, anti PD-L1, anti CTLA4) has led to fast and long lived responses in cancer patients. Response rates however are low and new treatments that enhance these rates are needed. Recent studies have shown that the administration of immune checkpoint blockers is associated with the overexpression of indoleamine 2,3-dioxygenase 1 (IDO1). The resulting immunoregulatory phenotype counteracts immune checkpoint blockade and allows for cancer progression. The discovery of IDO1 inhibitors, and the potential to combine them with immune checkpoint blockers, therefore represents an attractive strategy to fight cancer. We carried out a ligand-based virtual screen with > 1,000,000 commercially available small molecules. In vitro screening of the resulting 610 virtual hits provided us with 2 IDO1-selective, 2 TDO2-selective and 2 IDO1/TDO2-dual confirmed hits. (TDO2 is a protein with similar biochemical activity to IDO1 that is essential to tryptophan homeostasis.) A subsequent Hit to Lead campaign led to the identification of novel chemotypes that display potency similar or superior to IDO1 inhibitors currently under clinical investigation in IFN-γ stimulated (i.e. IDO1+) HeLa cells, with no sign of cytotoxicity. We have demonstrated that these compounds are > 1000-fold selective for IDO1 over TDO2 using cellular assays. IDO1 upregulation by cancer cells is known to be one of the mechanisms by which cancer cells evade the immune system. In an in vitro co-culture assay of cancer cells and T cells we have demonstrated our compounds can rescue T cell proliferation with EC50 values between 10 and 50 nM. We have also demonstrated that our compounds inhibit IDO1 in monocyte derived human dendritic cells. Interestingly, despite this potent cellular activity demonstrated in multiple disease relevant cellular assays, this chemotype failed to inhibit recombinant IDO1 in an isolated biochemical assay performed under reducing conditions, whereas the reference compound epacadostat provided activity comparable to literature values. In order to confirm our cellular effects were due to direct inhibition of IDO1 we set up thermal shift assays. Thermal shift assays using purified IDO1 protein have demonstrated that our compounds directly bind IDO1, and cellular thermal shift assays have confirmed direct target engagement in intact cells (stimulated for IDO1 expression). These compounds have physicochemical properties that would support oral dosing and display low in vitro CYP450 and hERG inhibition, thus reducing the risk of toxicity in the clinic. Our IDO1 inhibitors show a novel differentiated mode of action at the cellular level, and the consequences of this profile in terms of in vivo characterisation is ongoing. Citation Format: Thomas Pesnot, Sachin Mahale, Philip MacFaul, John Maclean, Caroline Phillips, Matilda Bingham, Catherine Eagle, James Kelly, Abhijith Thippeswamy, Simon Armitage, Aleksandr Grisin, Sheenagh Aiken, Lucy Cartwright, Richard Armer. Development of 2nd generation indoleamine 2,3-dioxygenase 1 (IDO1) selective inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5581. doi:10.1158/1538-7445.AM2017-5581

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile.

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

IRAK4 plays a critical role in the IL-1R and TLR signalling, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of permeable N-(1H-pyrazol-4-yl)carboxamides was developed by introducing lipophilic bicyclic cores in place of the polar pyrazolopyrimidine core of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides. Replacement of the pyrazolo[1,5-a]pyrimidine core with the pyrrolo[2,1-f][1,2,4]triazine, the pyrrolo[1,2-b]pyridazine, and thieno[2,3-b]pyrazine cores guided by cLogD led to the identification of highly permeable IRAK4 inhibitors with excellent potency and kinase selectivity.