Despite recent advances in the inhibition of EGFR (epidermal growth factor receptor), there remains a clinical need for new EGFR Exon20 insertion (Ex20Ins) inhibitors that spare EGFR WT. Herein, we report the discovery and optimization of two chemical series leading to ether 23 and biaryl 36 as potent, selective, and brain-penetrant inhibitors of Ex20Ins mutants. Building on our earlier discovery of alkyne 5 which allowed access to CNS property space for an Ex20Ins inhibitor, we utilized structure-based design to move to lower lipophilicity and lower CLint compounds while maintaining a WT selectivity margin. During optimization, aldehyde oxidase (AO) metabolism was identified as a human clearance risk, and through SAR exploration, lower AO metabolism was achieved. Potency and WT margin were optimized across a range of Ex20Ins mutants including the potential acquired resistance T790M mutant and efficacy demonstrated in an LXF2478 Ex20Ins ASV model with margin to EGFR WT in vivo.

Herein, we report the identification and optimization of a series of potent inhibitors of EGFR Exon20 insertions with significant selectivity over wild-type EGFR. A strategically designed HTS campaign, multiple iterations of structure-based drug design (SBDD), and tactical linker replacement led to a potent and wild-type selective series of molecules and ultimately the discovery of 36. Compound 36 is a potent and selective inhibitor of EGFR Exon20 insertions and has demonstrated encouraging efficacy in NSCLC EGFR CRISPR-engineered H2073 xenografts that carry an SVD Exon20 insertion and reduced efficacy in a H2073 wild-type EGFR xenograft model compared to CLN-081 (5), indicating that 36 may have lower EGFR wild-type associated toxicity.

To further facilitate the discovery of cysteine reactive covalent inhibitors, there is a need to develop new reactive groups beyond the traditional acrylamide-type warheads. Herein we describe the design and synthesis of covalent EGFR inhibitors that use vinylpyridine as the reactive group. Structure-based design identified the quinazoline-containing vinylpyridine 6 as a starting point. Further modifications focused on reducing reactivity resulted in substituted vinyl compound 12, which shows high EGFR potency and good kinase selectivity, as well as significantly reduced reactivity compared to the starting compound 6, confirming that vinylpyridines can be applied as an alternative cysteine reactive warhead with tunable reactivity.

PARP inhibitors have attracted considerable interest in drug discovery due to the clinical success of first-generation agents such as olaparib, niraparib, rucaparib, and talazoparib. Their success lies in their ability to trap PARP to DNA; however, first-generation PARP inhibitors were not strictly optimized for trapping nor for selectivity among the PARP enzyme family. Previously we described the discovery of the second-generation PARP inhibitor AZD5305, a selective PARP1-DNA trapper. AZD5305 maintained the antitumor efficacy of first-generation PARP inhibitors while exhibiting lower hematological toxicity. Recently, there has been interest in central nervous system (CNS)-penetrant PARP inhibitors for CNS malignancies and other neurological conditions; however, AZD5305 is not CNS penetrant. Herein we describe the discovery and optimization of a series of CNS-penetrant, PARP1-selective inhibitors and PARP1-DNA trappers, culminating in the discovery of AZD9574, a compound that maintains the PARP1 selectivity of AZD5305 with improved permeability, reduced efflux, and increased CNS penetration.

Supplementary Figure 1: Osimertinib monotherapy induce tumor growth inhibition in a NSCLC EGFR G719A;S768I mutation PDX model in vivo. Supplementary Figure 2: Osimertinib as monotherapy induce a stronger inhibition of the level of pEGFR and the downstream signalling pathways than afatinib. Supplementary Figure 3: MET amplification induces resistance to osimertinib treatment in a NSCLC EGFR G719A mutation PDX model in vivo.

Supplementary figure 1: Generation of H2073 ASV and H2073 SVD. Supplementary figure 2: H2073 parental and Ex20Ins are dependent on EGFR for survival in vitro. Supplementary figure 3: Effect of TKI on the Sytox Proliferation of H2073 wt, H2073-SVD and H2073-ASV, in vitro. Supplementary figure 4: The H2073 parental NSCLC xenograft model is sensitive to afatinib in vivo. Supplementary figure 5: Osimertinib and AZ5104 given as monotherapy induce robust inhibition of p-EGFR and relevant downstream signalling pathways. Supplementary figure 6: Osimertinib and AZ5104 as monotherapy induce a strong inhibition of the level of pEGFR and the downstream signalling pathways. Supplementary figure 7: Osimertinib, AZ5104, afatinib and erlotinib are well tolerated at the dose explored in the PDX models and AZ5104 induce significant tumor growth inhibition in the LU0387 model when dosed at 50 mg/kg. Supplementary figure 8: Osimertinib, afatinib and erlotinib robustly inhibit pEGFR; however, select proteins representing downstream signalling pathways (pAKT) have enhanced inhibition to osimertinib and pS6R remains suppressed at early timepoints compared to afatinib and erlotinib in the UC Davis PDX model harboring the V769_D770InsASV.

The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.

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.

Abstract Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M–resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non–small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent. Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I. Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.

Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity.A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab.EGFR D770-N771InsSVD cell phospho IC50 (µM)EGFR WT (H2073) cell phospho IC50 (µM)Fold-EGFR WT margin (cell)afatinib0.0060.00330.5osimertinib0.0940.414.4poziotinib0.00340.00351TAS64170.0230.0672.9AZ62810.0972.223Citation Format: Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc’h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4813.

Osimertinib is a next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with activity against both the activating and the ‘gatekeeper’ T790M EGFR mutations. An acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in approximately 15% and 7% of patients in second-line and first-line treatment respectively. This percentage in the first-line setting will likely evolve as the first line data mature. The C797S mutation leads to the loss of covalent binding of osimertinib to mutant EGFR. The high affinity of the EGFR triple mutant for ATP presents a challenge for reversible inhibitor design, particularly as the loss of the cysteine at position 797 precludes the previously exploited covalent approaches. We have explored various approaches to address this challenge, including an effort to maximise reversible affinity to target the C797S mutation without requiring a covalent bond. We describe herein the therapeutic potential of reversible phosphine oxide pyrazole inhibitors in tumors harboring C797S. Using structure-based design, we were able to design a series of phosphine oxide pyrazole inhibitors that displayed exceptionally high biochemical potency against EGFR C797S mutation, which translated into good activity in cell-based assays. Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease-relevant models to express the C797S mutation to evaluate potency in vitro and in vivo. By modulating the physicochemical properties of our in vitro leads, we were able to achieve good oral exposure of cellularly active EGFR C797S inhibitors such as AZ’7608. We showed that AZ’7608 inhibits signalling pathways and cellular growth of C797S EGFR cell lines in vitro and demonstrated an improved WT EGFR margin. This translated into 52% (p The work presented herein shows a proof of concept for reversible phosphine oxide pyrazole inhibitors to target tumors harboring C797S. The emergence of the C797S EGFR mutation remains a key area of unmet need and warrants further efforts in drug discovery. Citation Format: Nicolas Floch, M. Raymond V. Finlay, Ambra Bianco, Sue Bickerton, Nicola Colclough, Darren A. Cross, Emanuela M. Cuomo, Carine M. Guerot, David Hargreaves, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Amar Rahi, Paul D. Smith, Richard A. Ward. Evaluation of the therapeutic potential of phosphine oxide pyrazole inhibitors in tumors harboring EGFR C797S mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4451.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

Abstract Four poly(ADP-ribose) polymerase (PARP) inhibitors have now presented phase 3 monotherapy data showing compelling benefit of targeting tumours enriched with DNA damage response (DDR) pathway deficiencies, including BRCA gene mutations. Indirect treatment comparisons using the published clinical data from these late stage trials suggest similar levels of monotherapy efficacy are observed in spite of reported differences in PARP trapping potency. However, there is greater diversity in the observed safety profiles. To try and understand these observations, we have carried out a head-to-head comparison of these four PARP inhibitors (olaparib, niraparib, rucaparib and talazoparib) as well as veliparib, which recently reported phase 3 chemotherapy combination data. In our studies, we included an assessment of molecular mechanism of action that included PAR inhibition, PARP trapping and synthetic lethality in isogenic BRCA mutant and wild type models. In addition, an assessment of selectivity in terms of both inhibition of PARP family members using a novel chemoproteomic approach, as well as secondary (off-target) activities was performed. Finally, effects on human haematopoietic stem cell viability and bio-distribution to bone marrow in the rat were tested and compared. A detailed correlation of our datasets with the observed clinical results, including adverse events, suggests these preclinical experiments provide an excellent predictor of clinical response and could be used to assess emerging as well as novel PARP inhibitors. OlaparibVeliparibRucaparibNiraparibTalazoparibCompanyAZAbbVieClovisTesaroPfizerPhaseApprovedIIIApprovedApprovedIIIPARP1 SPR Kd (µM)0.0010.0070.0010.0130.002PARP2 SPR Kd (µM)0.0010.0140.0230.0430.005PARPs with Proteomic Kd <1 µM1,2,3,4,131,2,3,4,131,2,3,4,10,131,2,131,2,3,4,5a,13, 16Sec. Pharm. #, top hit µM0/855/85 5HT7, 0.513/85 5HT4, 0.517/84 DAT, 0.040/85Monotherapy dose (mg)300 bd (tablet)500 bd600 bd300 od1 od Citation Format: Elisabetta Leo, Jeffrey Johannes, Giuditta Illuzzi, Andrew Zhang, Paul Hemsley, Michal J. Bista, Jonathan P. Orme, Verity A. Talbot, Ana J. Narvaez, Elizabeth Underwood, Andrew Pike, Jenni K. Nikkila, Lucy Riches, Sinbad Sweeney, Frida Gustafsson, Anna Cronin, Piero Ricchiuto, Debora A. Roaquin, Fiona Pachl, Eric Miele, Ruth MacDonald, Glen Hawthorne, Andrew N. Mead, Mark J. O'Connor. A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-273.

Abstract EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non–small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo. The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885–96. ©2018 AACR.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

This report describes the implementation of an automated work cell with commercially available hardware and software, capable of handling up to 15 separate reagents for performing 96-well or 384-well assays but with a small footprint and only a single liquid dispenser and two plate washers. Extremely flexible software was used to enable this simple work cell to perform processes that would traditionally require a much larger, more expensive automation platform. With the development of the C-Myc assays for the targets DYRK, BMX, PERK, and FAK, the authors describe a software solution to multibatch assays to run simultaneously, reducing reagent dead volume and increasing the efficiency of running multiple assays such that the time to generate data across multiple targets was significantly shortened. Although a larger automated system with multiple robotic arms and extensive equipment would also be able to process multiple assays simultaneously, the work cell we have described represents an inexpensive and flexible, easily upgradable option suitable for a wider range of labs. (Journal of Biomolecular Screening. 2011;16:967-973)

This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a generic enzyme-linked immunosorbent assay method was designed that introduced a c-Myc tag to four different targets and allowed all four cell assays to be run using a standardized process. These process improvements yielded cost savings and greatly reduced the required resource, as well as reducing timelines for developing cellular assays. (Journal of Biomolecular Screening 2011; 16: 959-966)