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The structure-based design of small-molecule inhibitors targeting protein–protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

In this article, we report the discovery of a series of pyrimidopyridones as inhibitors of IRAK4 kinase. From a previously disclosed 5-azaquinazoline series, we found that switching the pyridine ring for an N-substituted pyridone gave a novel hinge binding scaffold which retained potency against IRAK4. Importantly, introduction of the carbonyl established an internal hydrogen bond with the 4-NH, establishing a conformational lock and allowing truncation of the large basic substituent to a 1-methylcyclopyl group. Subsequent optimisation, facilitated by X-ray crystal structures, allowed identification of preferred substituents at both the pyridone core and pyrazole. Subsequent combinations of optimal groups allowed control of lipophilicity and identification of potent and selective inhibitors of IRAK4 with better in vitro permeability and lower clearance.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.

Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.

The current clinically approved PARP inhibitors have limited subtype selectivity and are to some degree restricted in their ability to penetrate the central nervous system (CNS) due to efflux transporters, potentially limiting their efficacy in treating metastatic disease or primary tumors in the brain. The current study evaluated the potential of AZD9574, a next generation, PARP1 selective inhibitor/trapper, to penetrate the CNS in cynomolgus monkey, and its occupancy of the PARP1 enzyme, using positron emission tomography (PET). In vitro bidirectional efflux assay data suggested AZD9574 showed minimal substrate potential compared to the clinically approved PARP inhibitors. This was reflected in an increased ratio of unbound brain to unbound plasma concentration (Kpuu) in the rat of ~0.31. Therefore AZD9574 was taken forward into cynomolgus monkey PET studies. Firstly, the Kpuu was determined following dosing of [11C]AZD9574, co-administered with unlabeled drug to minimize the impact of specific binding. The high specific signal observed lead to the development of [11C]AZ3391, a PARP1 selective, CNS penetrant PET tracer, which was subsequently used to directly assess the PARP1 target engagement of AZD9574 in the brain. AZD9574 was found to show a Kpuu in cynomolgus monkeys of 0.79, close to unity with unbound plasma concentrations suggesting minimal CNS restriction. Furthermore, an i.v. infusion dose response study with AZD9574, conducted to examine its ability to block target occupancy by the PET tracer [11C]AZ3391, demonstrated a reduction in [11C]AZ3391 accumulation in whole brain. The resulting calculated occupancy of AZD9574 ranged from 17% for the lowest dose (0.003 mg/kg) to 95% for the highest dose tested (0.05 mg/kg). Comparable reduction in occupancy was seen for peripheral tissue, such as bone marrow, supporting the conclusion that AZD9574 shows minimal CNS restriction. These data show that AZD9574 is the first PARP inhibitor to reach the clinic which combines PARP1 selectivity, trapping and high CNS penetration in a single molecule and supports its development as a potential therapy for the treatment of metastatic disease and primary brain tumors. Citation Format: Andy Pike, Amber Balazs, Zsolt Cselényi, Sébastien L. Degorce, Avipsa Ghosh, Sudhir M. Hande, Jeffrey Johannes, Peter Johnström, Martin J. Packer, Magnus Schou, XiaoLan Zheng. Evaluation of the CNS penetration of a next generation PARP inhibitor, AZD9574, in cynomolgus monkey using positron emission tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5076.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

We report the first disclosure of IRAK3 degraders in the scientific literature. Taking advantage of an opportune byproduct obtained during our efforts to identify IRAK4 inhibitors, we identified ready-to-use, selective IRAK3 ligands in our compound collection with the required properties for conversion into proteolysis-targeting chimera (PROTAC) degraders. This work culminated with the discovery of PROTAC 23, which we demonstrated to be a potent and selective degrader of IRAK3 after 16 h in THP1 cells. 23 induced proteasome-dependent degradation of IRAK3 and required both CRBN and IRAK3 binding for activity. We conclude that PROTAC 23 constitutes an excellent in vitro tool with which to interrogate the biology of IRAK3.

In this article, we report our investigation of a phenomenon by which bridging morpholines across the ring with one-carbon tethers leads to a counterintuitive reduction in lipophilicity. This effect was also found to occur in piperazines and piperidines and lowered the measured log D7.4 of the bridged molecules by as much as −0.8 relative to their unbridged counterparts. As lowering lipophilicity without introducing additional heteroatoms can be desirable, we believe this potentially provides a useful tactic to improve the drug-like properties of molecules containing morpholine-, piperazine-, and piperidine-like motifs.

ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

Since the approval of olaparib in 2014 for BRCA mutated (BRCAm) ovarian cancer, many PARP inhibitors have been developed and have seen widespread success. However, as a class, these drugs are not without adverse events which have limited their ability to be combined with chemotherapy. Most first generation PARP inhibitors were developed and optimized before the concept of PARP1-DNA trapping was discovered as the mechanism by which PARP inhibitors exert their synthetic lethal effects on BRCAm cells. Moreover, the first generation PARP inhibitors were not optimized for selectivity across the PARP family potentially driving undesirable side effects, including intestinal toxicity from tankyrase inhibition or hematological toxicity from PARP2 inhibition. With this in mind, we set out to discover a best-in-class, second generation PARP inhibitor that was highly selective for PARP1 over the other 16 members of the PARP family, as well as a highly potent PARP1-DNA trapper. PARP1 and PARP2 have a highly similar amino acid sequence, and most of the residues around the nicotinamide binding site are identical. However, there are some key residue differences in the helical domain which serves a regulator of the nicotinamide binding pocket. The publication of NMS-P118 in 2015 by Nerviano Medical Sciences showed that a highly selective PARP1 inhibitor could be found. This work inspired us to screen an extensive list of previously reported PARP inhibitors for selectivity against PARP2 and we found that FR257516 met the selectivity criteria as previously reported, but lacked the ability to trap PARP1 to DNA and hence lacked any activity in a cell colony formation assay in DLD-1 BRCA2-/- cells. Using parallel chemistry to generate diverse analogs, X-ray crystallography to enable structure-based design, and exploration of multiple nicotinamide mimetic cores, we were able to generate lead compound AZ4554, which was a PARP1 selective PARP1-DNA trapper with potent activity in BRCAm cells. Using concepts of property-based drug design, we were able to optimize lead compound AZ4554 into candidate drug AZD5305, making key improvements in secondary pharmacology, including reducing hERG activity, and intrinsic clearance in human microsomes through the introduction of polar atoms to lower logD without compromising permeability or oral bioavailability. AZD5305 is a highly selective binder of PARP1 over PARP2 and other PARP enzymes by fluorescence polarization, surface plasmon resonance, and single molecule spectroscopy. It is highly potent against DLD-1 BRCA2-/- cells, while sparing isogenic BRCA WT cells. The secondary pharmacology of AZD5305 is remarkably clean, with hERG activity >40 µM. AZD5305 has a very favorable pre-clinical PK profile, low predicted human dose, and has shown efficacy in an MDA-MB-436 mouse xenograft model. Citation Format: Sudhir Hande, Amber Balazs, Sébastien L. Degorce, Kevin Embrey, Avipsa Ghosh, Sonja J. Gill, Anders Gunnarsson, Giuditta Illuzzi, Jordan Lane, Carrie Larner, Elisabetta Leo, Andrew Madin, Lisa McWilliams, Mark J. O'Connor, Jonathan Orme, Fiona Pachl, Martin Packer, Andy Pike, Philip Rawlins, Marianne Schimpl, Anna D. Staniszewska, Andrew Zhang, Xiaolan Zheng, Jeffrey W. Johannes. Structure-based and property-based drug design of AZD5305, a highly selective PARP1 inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 296.

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline

[Image: see text] We have conducted an analysis of azaspiro[3.3]heptanes used as replacements for morpholines, piperidines, and piperazines in a medicinal chemistry context. In most cases, introducing a spirocyclic center lowered the measured logD(7.4) of the corresponding molecules by as much as −1.0 relative to the more usual heterocycle. This may seem counterintuitive, as the net change in the molecule is the addition of a single carbon atom, but it may be rationalized in terms of increased basicity. An exception to this was found with N-linked 2-azaspiro[3.3]heptane, where logD(7.4) increased by as much as +0.5, consistent with the addition of carbon. During our investigation, we also concluded that azaspiro[3.3]heptanes are most likely not suitable bioisosteres for morpholines, piperidines, and piperazines, when not used as terminal groups, due to significant changes in their geometry.

A novel series of 3-quinoline carboxamides has been discovered and optimized as selective inhibitors of the ataxia telangiectasia mutated (ATM) kinase. From a modestly potent HTS hit (4), we identified molecules such as 6-[6-(methoxymethyl)-3-pyridinyl]-4-{[(1R)-1-(tetrahydro-2H-pyran-4-yl)ethyl]amino}-3-quinolinecarboxamide (72) and 7-fluoro-6-[6-(methoxymethyl)pyridin-3-yl]-4-{[(1S)-1-(1-methyl-1H-pyrazol-3-yl)ethyl]amino}quinoline-3-carboxamide (74) as potent and highly selective ATM inhibitors with overall ADME properties suitable for oral administration. 72 and 74 constitute excellent oral tools to probe ATM inhibition in vivo. Efficacy in combination with the DSB-inducing agent irinotecan was observed in a disease relevant model.

We report the discovery and optimisation of a series of 8-(2,3-dihydro-1,4-benzoxazin-4-ylmethyl)-2-morpholino-4-oxo-chromene-6-carboxamides, leading to compound 16 as a potent and selective PI3Kβ/δ inhibitor: PI3Kβ cell IC50 0.012 μM (in PTEN null MDA-MB-468 cell) and PI3Kδ cell IC50 0.047 μM (in Jeko-1 B-cell), with good pharmacokinetics and physical properties. In vivo, 16 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-deficient PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Compound 16 was selected as a preclinical candidate for the treatment of PTEN-deficient tumours.

Optimization of cellular lipophilic ligand efficiency (LLE) in a series of 2-anilino-pyrimidine IGF-1R kinase inhibitors led to the identification of novel 2-(pyrazol-4-ylamino)-pyrimidines with improved physicochemical properties. Replacement of the imidazo[1,2-a]pyridine group of the previously reported inhibitor 3 with the related pyrazolo[1,5-a]pyridine improved IGF-1R cellular potency. Substitution of the amino-pyrazole group was key to obtaining excellent kinase selectivity and pharmacokinetic parameters suitable for oral dosing, which led to the discovery of (2R)-1-[4-(4-{[5-chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)-2-pyrimidinyl]amino}-3,5-dimethyl-1H-pyrazol-1-yl)-1-piperidinyl]-2-hydroxy-1-propanone (AZD9362, 28), a novel, efficacious inhibitor of IGF-1R.

We have developed a series of orally efficacious IRAK4 inhibitors, based on a scaffold hopping strategy and using rational structure based design. Efforts to tackle low permeability and high efflux in our previously reported pyrrolopyrimidine series (Scott et al., 2017) led to the identification of pyrrolotriazines which contained one less formal hydrogen bond donor and were intrinsically more lipophilic. Further optimisation of substituents on this pyrrolotriazine core culminated with the discovery of 30 as a promising in vivo probe to assess the potential of IRAK4 inhibition for the treatment of MyD88 mutant DLBCL in combination with a BTK inhibitor. When tested in an ABC-DLBCL model with a dual MyD88/CD79 mutation (OCI-LY10), 30 demonstrated tumour regressions in combination with ibrutinib.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88

A growing number of early discovery collaborative agreements are being put in place between large pharma companies and partners in which the rights for assets can reside with a partner, exclusively or jointly. Our corporate screening collection, like many others, was built on the premise that compounds generated in-house and not the subject of paper or patent disclosure were proprietary to the company. Collaborative screening arrangements and medicinal chemistry now make the origin, ownership rights and usage of compounds difficult to determine and manage. The Compound Passport Service is a dynamic database, managed and accessed through a set of reusable services that borrows from social media concepts to allow sample owners to take control of their samples in a much more active way.

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

Several studies have highlighted the dependency of PTEN deficient tumors to PI3Kβ activity and specific inhibition of PI3Kδ has been shown activity against human B-cell cancers. We describe the discovery and optimization of a series of 8-(1-anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kβ/δ inhibitors, which led to the discovery of the clinical candidate 13, also known as AZD8186. On the basis of the lower lipophilicity of the chromen-4-one core compared to the previously utilized pyrido[1,2-a]pyrimid-4-one core, this series of compounds displayed high metabolic stability and suitable physical properties for oral administration. Compound 13 showed profound pharmacodynamic modulation of p-Akt in PTEN-deficient PC3 prostate tumor bearing mice after oral administration and showed complete inhibition of tumor growth in the mouse PTEN-deficient PC3 prostate tumor xenograft model. 13 was selected as a clinical candidate for treatment of PTEN-deficient cancers and has recently entered phase I clinical trials.

Herein we describe the synthesis of a series of novel, functionalised pyrrolothiazolo–pyrimidines, –pyridines and –pyrazines employing a short reaction sequence, utilising the under-reported interrupted Pummerer reaction to effect cyclisation in the final step.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

Attempts to lock the active conformation of compound 4, a PI3Kβ/δ inhibitor (PI3Kβ cell IC50 0.015 μM), led to the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-ones, which showed high levels of potency and selectivity as PI3Kβ/δ inhibitors. Compound 10 proved exquisitely potent and selective: PI3Kβ cell IC50 0.0011 μM in PTEN null MDA-MB-468 cell and PI3Kδ cell IC50 0.014 μM in Jeko-1 B-cell, and exhibited suitable physical properties for oral administration. In vivo, compound 10 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-null PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Based on these results, compound 10 was selected as one of our PI3Kβ/δ preclinical candidates.

To gain insights into the tumor microenvironment in primary and secondary CNS lymphomas, we applied LC/MS and GC/MS for differential metabolomic profiling of the cerebrospinal fluid (CSF) of CNS lymphoma patients compared to control subjects. Among 145 analytes identified, the majority were involved in energy metabolism; one of the most significantly upregulated metabolites in CNS lymphoma was lactate (1.8 fold, p Thus far we have applied these metabolic imaging approaches to preclinical murine models involving diffusely invasive, intracranial, patient-derived xenografts of CNS DLBCL in RAG-/- mice, to detect tumor-associated lactate production generated by infiltrating lymphoma. We demonstrated that each of these MRS approaches detect highly invasive lymphoma that is undetectable by conventional gadolinium-enhanced T1, T2 sequences, or diffusion-weighted imaging. Because of its ability to detect real-time changes in metabolic pathways, we focused on the application of HP13C pyruvate metabolic imaging as a non-invasive imaging tool for NF-kB pathway inhibition in CNS lymphoma using these models. We evaluated the metabolic response to AZ1495, a novel, CNS penetrant, orally-bioavailable inhibitor of IRAK4 kinase, comparing MYD88 wild type vs. MYD88 L265P mutant tumor models. Using a 14.1T imaging system for MR acquisition, we demonstrated similar tumor-associated production of HP 13C lactate in both MYD88wt and MY88 mutant tumors at 3 weeks post-implantation. We determined that while AZ1495 did not significantly impact lactate production in MYD88wt lymphoma, this agent significantly down-regulated tumor-associated HP pyruvate to lactate conversion (>47%) within 2 days in MYD88 mutant CNS lymphoma (p 2-fold down-modulation of NF-kB gene expression at 4h of AZ1495 therapy, including transcripts encoding LDH-A as well as the catalytic subunit of PI3K, suggesting interaction with the B cell receptor pathway. Combination AZ1495 plus ibrutinib starting d+5 was synergistic in survival prolongation compared to AZ1495 monotherapy (p Disclosures Gao: Glaxo Smith Kline: Employment. Drew:AstraZeneca: Employment. Degorce:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Anjum:Astra Zeneca: Employment. Bloecher:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.

In primary central nervous system lymphoma (PCNSL), L265P mutation of the myeloid differentiation 88 (MYD88) protein is the most common, and induces a constitutive activation of IRAK4 kinase, IRAK1 phosphorylation and increased NF-kB signaling, offering a unique window for therapeutic development. We previously show that AZ1495, a potent inhibitor of IRAK4, significantly delayed progression and improved OS in L265P-mutated PCNSL models [ASH 2016], highlighting the clinical potential of IRAK inhibition. In the clinic however, early monitoring of such targeted therapies remains challenging using conventional imaging, as treatment often results in non-readily detectable anatomical changes. Here, we questioned the potential of a new neuroimaging method, hyperpolarized 13C magnetic resonance spectroscopic imaging (HP-13C MRSI), to non-invasively monitor IRAK inhibition in PCNSL for the first time. Two PCNSL patient-derived xenografts models were studied: one harboring the L265P MYD88 mutation (MYD88mut), and the other, wild type for this protein (MYD88wt). Using HP-13C MRSI, a high production of HP [1-13C] lactate was detected in both MYD88wt and MYD88mut (0.5 ± 0.04, n=2 MYD88wt; 0.61 ± 0.2, n=4 MYD88mut), indicating PCNSL presence throughout the brain. At day 2 and 4 of AZ1495 treatment, the HP [1-13C] lactate-to-pyruvate ratio remains unchanged in MYD88wt animals at all treatment time points (p=0.29 and p=0.29 at day 2 and 4 when compared to baseline, respectively) On the other hand, this ratio significantly decreased in MYD88mut animals at all time points (36.6% at day 2, p=0.07 and 56.3% at day 4, p=0.0006), in line with the previously reported effect of AZ1495 treatment on MYD88mut PCNSL only. To conlude, we showed that HP-13C MRSI can detect MYD88-mutation-specific modulations of HP lactate production following IRAK inhibition. Because HP-13C MRSI is clinically translatable and expanding rapidly, this study is of high significance for future clinical trials, to enhance monitoring of PCNSL progression and response of targeted therapies.

A series of tetrahydroisoquinoline phenols was modified to give an estrogen receptor downregulator-antagonist profile. Optimization around the core, alkyl side chain, and pendant aryl ring resulted in compounds with subnanomolar levels of potency. The phenol functionality was shown to be required to achieve highly potent compounds, but unusually this was compatible with obtaining high oral bioavailabilities in rat.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.

Among the clinical subtypes of diffuse large B-cell lymphoma, central nervous system (CNS) lymphomas, both primary (PCNSL) and secondary (SCNSL), are generally associated with the worst prognosis. Moreover, PCNSL typically exhibits multifocal dissemination, prompting its description as a whole brain disease. An accumulation of genomic data suggests that aberrant NF-kB pro-survival signaling may be a critical factor in the pathogenesis of CNS lymphoma. To date, however, there has been an absence of mechanistic studies to interrogate the mediators and significance of NF-kB activation in CNS lymphoma invasion. Recently our lab derived the first panel of xenografts yet established from PCNSL patients (n=7), and defined their invasive properties in murine models. These novel model systems provide a unique opportunity to study key regulatory mechanisms as well as novel targeted therapies of CNS lymphoma invasion and growth within the brain. We determined that the intracranial growth patterns of the xenografts can be classified based upon reproducible patterns of invasiveness. Two xenografts derived from SCNSL failed to infiltrate brain parenchyma or to migrate to the contralateral hemisphere; instead these tumors exhibited solely a meningeal dissemination pattern, largely growing in the ventricles. By contrast, five of the xenografts exhibited diffuse brain invasion into bilateral hemispheres. In addition, we noted that CNS lymphoma xenografts with an invasive phenotype can be sub-divided phenotypically into moderately vs. highly invasive tumors. After standard injection of 200,000 tumor cells/brain, the highly invasive lymphomas were reproducibly lethal to mice within 3 weeks post-implantation and yielded ~ 1X106 lymphoma cells/brain at necropsy. Under identical conditions, the moderately-invasive lymphoma xenografts were lethal to mice within 1-3 months and yielded between 3-5 X106lymphoma cells/brain. Notably each of the highly invasive CNS lymphoma xenografts were derived from CNS lymphomas that contained the activating L265P mutation of MYD88 (activator of NF-kB via IRAK1/4) and/or Y196H mutation of CD79B (activator of NF-kB), whereas the non-invasive xenografts contained wild type MYD88 and CD79B. We analyzed RNA-seq expression data of these xenograft samples to evaluate relationship of individual genes and signaling pathways with the distinct subclasses of CNS lymphomas in terms of invasiveness. Upregulated signatures in the pro-invasive phenotype are significantly enriched with NF-kB target genes (http://lymphochip.nih.gov/signaturedb/) (FDR Given that the L265P mutation of MYD88 represents a candidate key driver of NF-kB activation in CNS lymphoma, we evaluated the preclinical efficacy of AZ1495, a novel and potent inhibitor of IRAKs. We demonstrated CNS penetration of AZ1495 after PO administration to mice with intracranial lymphoma xenografts, yielding a mean brain/plasma partition coefficient of 0.8. AZ1495 potently antagonized NF-kB activation as demonstrated by p-p65 immunoblot analysis of tumor lysates isolated from murine CNS lymphoma xenografts, within 3 hrs of administration. AZ1495 monotherapy resulted in > 2-fold delay in both intracranial plus spinal progression in a dissemination model of CNS lymphoma using L265P mutant OCI-LY10 cells engineered for bioluminescence (p2-fold increase in median OS in a patient-derived model of L265P mutant PCNSL. These results support the preclinical utility of these models to dissect impact of NF-kB inhibition on CNS lymphoma progression as well as the clinical potential of IRAK antagonism as a means to target NF-kB activation in L265P mutant CNS lymphomas. Disclosures Anjum: Astra Zeneca: Employment. Drew:Astra Zeneca: Employment. Degorce:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.