The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.

The structure-based design of small-molecule inhibitors targeting protein-protein inter-actions (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 remarka-ble 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 conforma-tion. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells via PARP1 catalytic inhibition and PARP1 trapping onto the DNA. All known clinical PARP1 inhibitors bind at the same site at the catalytic center of the enzyme. However, despite this resemblance they show immensely different outcomes in terms of response rate in the clinic due to their varying degree of PARP trapping ability. 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. There has been strong rationale for the use of PARP inhibitors in neuro-oncology. However, the first-generation PARP inhibitors have limited CNS distribution as these drugs were not designed for brain penetration. Recently AstraZeneca has reported the discovery of AZD5305, a next generation PARP1 selective inhibitor and PARP1-DNA trapper which was not designed with a CNS penetrant profile. Given the unmet need of a brain penetrant PARP1 inhibitor, we set out to identify a highly potent and selective PARP1 inhibitor and trapper with CNS profile. In our next generation PARP1 inhibitor, we sought to retain the profile of AZD5305 and lower the efflux for CNS penetration. Despite the challenge of narrow SAR, we successfully used the structure- and property-based design approach to identify a brain penetrant PARP1 inhibitor and PARP1-DNA trapper. We used multiple medicinal chemistry maneuvers such as masking the hydrogen bond donors and core modifications to lower the efflux in order to achieve brain penetration. Further optimization of the nicotinamide mimetic core for potency and metabolic stability led us to the discovery of AZD9574.AZD9574 shows improved selectivity for PARP1 over PARP2 vs AZD5305 and retains its excellent selectivity over other PARP family members. It has low efflux in Caco2, MDCK-MDR1, and MDCK-MDR1-BCRP permeability assays and it also showed CNS penetration in rat and cynomolgus monkey. AZD9574 has excellent secondary pharmacology and acceptable physicochemical properties and good PK in preclinical species.In vitro, AZD9574 selectively inhibits the growth of BRCAm cell lines. Importantly, AZD9574 showed efficacy in an intracranial BRCA1m MDA-MB-436 xenograft model at doses of 3, 10 and 30 mg/kg QD, significantly extending the survival of tumor-bearing mice compared to vehicle control arm.In summary, AZD9574 is a next generation selective PARP1 inhibitor and trapper with CNS penetration. This profile makes it an ideal candidate for treating CNS malignancies or brain metastases that have a dependence on PARP inhibition either as single agent or in combination with other therapies. Citation Format: Avipsa Ghosh, Sudhir M. Hande, Amber Balazs, Derek Barratt, Sabina Cosulich, Barry Davies, Sébastien Degorce, Kevin Embrey, Sonja Gill, Anders Gunnarsson, Giuditta Illuzzi, Peter Johnström, Jordan Lane, Carrie Larner, Rachel Lawrence, Elisabetta Leo, Andrew Madin, Elizabeth Martin, Lisa McWilliams, Lenka O’Connor, Mark O’Connor, Jonathan Orme, Fiona Pachl, Martin Packer, Andy Pike, Philip Rawlins, Marianne Schimpl, Magnus Schou, Anna Staniszewska, Wenzhan Yang, James Yates, Andrew Zhang, XiaoLa Zheng, Stephen Fawell, Petra Hamerlik, Jeffrey Johannes. Structure-based and property-based drug design of AZD9574, a CNS penetrant PARP1 selective inhibitor and trapper [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 6302.

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.

Abstract 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.

Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

The polycomb repressive complex 2(PRC2), consisting of the core subunits EED, EZH2, and SUZ12, is frequently overexpressed and deregulated in hematological and solid malignancies. Clinical validation for targeting the PRC2 complex has been demonstrated with several small molecule inhibitors of EED or EZH2 that are either approved or being evaluated in clinical trials in different cancer settings. Like other treatment modalities, single-agent activity is limited. Given that the PRC2 complex is involved in multiple diverse oncogenic/immune pathways, we thus explored the combination potential of inhibiting PRC2 in a wide array of biological contexts. To this end, we have developed a potent, selective, and orally bioavailable EED ligand. The compound binds to EED (K D =3.2 nM) and inhibits PRC2 enzyme activity (IC 50= 7nM). The compound inhibits proliferation of an EZH2 dependent Karpas422 GCB-DLBCL cell line (GI 50= 27nM) in vitro. In vivo, the inhibitor is well tolerated and drives tumor regression at 10 mpk and 50 mpk doses in a DLBCL xenograft model. Given the compound has favorable selectivity, potency and pharmacokinetic profile, we next evaluated the combinability of this EEDi with select compounds targeting diverse oncogenic pathways. We find that EED and PI3K/AKT inhibitors show marked combination activity in DLBCL, in part through downregulation of AKT signaling. Combination activity is also observed with the EED inhibitor in combination with the BTK inhibitor, acalabrutinib, in DLBCL, although this appears to be context dependent. In addition to DLBCL models, we have also assessed the therapeutic potential of EED inhibitor combinations in solid tumors. In SCLC, the EED inhibitor increased expression of SLFN11, a biomarker that has been linked to clinical response to a PARP inhibitor / temozolomide combination (Pietanza et al., 2018). Therefore, we evaluated the EED inhibitor in combination with the PARP inhibitor, olaparib, in an aggressive SCLC xenograft model and observed reduced tumor growth in the combination arm but not in monotherapy arms. In ovarian cancer, ARID1A mutations are proposed to sensitize to EZH2 and ATR inhibitors (Bitler et al., 2015, Williamson et al., 2016) which prompted evaluation of the EED inhibitor with the ATR inhibitor, AZD6738. Indeed, the combination resulted in tumor stasis whereas the monotherapies produced modest tumor growth inhibition. Last, we have evaluated the EED inhibitor alone, and in combination with a CTLA4 antibody, in a tumor immunity setting using an immunologically cold syngeneic melanoma model. Interestingly, we find that EEDi has significant anti-tumor activity alone, as compared with either control or CTLA4 antibody treatment alone. Together, these data show that while our EEDi has strong single-agent activity in vitro and in vivo, combining the EEDi together with inhibitors of select oncogenic pathways may bring deeper therapeutic response in a context dependent manner.

A key activity in small-molecule drug discovery is the characterization of compound-target interactions. Surface plasmon resonance (SPR) is a flexible technique for this purpose, with a wide affinity range (micromoles to picomoles), low protein requirements, and the ability to characterize the kinetics of compound binding. However, a key requirement of SPR is the immobilization of the target protein to the surface of the sensor chip. The most commonly used immobilization techniques (covalent immobilization, streptavidin-biotin) are irreversible in nature, which can afford excellent baseline stability but impose limitations throughput for slowly dissociating compounds or unstable targets. Reversible immobilization (e.g., His-tag-Ni-NTA) is possible but typically precludes accurate quantification of slow dissociation kinetics due to baseline drift.Here we present our investigation of three immobilization strategies (dual-His-tagged target protein, His-tagged streptavidin, and switchavidin) that combine the robustness of irreversible immobilization with the flexibility of reversible immobilization. Each has its own advantages and limitations, and while a universal immobilization procedure remains to be found, these strategies add to the immobilization toolbox that enables previously out-of-scope applications. Such applications are highlighted in two examples that greatly increased throughput for the kinetic characterization of potent kinase inhibitors and kinetic profiling of covalent inhibitors.

Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pK(D) similar to 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC(50) similar to 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI(50)] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here, we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor 'example 172' (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics, we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.

Abstract Deregulation of the PRC2 components EZH2, SUZ12, and EED plays critical roles in driving aberrant hypermethylation of H3K27 and tumorigenicity of many solid and hematological malignancies. Although SAM competitive small molecule inhibitors of EZH2 show promising clinical activity in PRC2-dependent cancers, preclinical data suggests that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (Proteolysis Targeting Chimera) to efficiently target EED for elimination. Our EED-targeting PROTACs bind to EED (pKD= 9.02-9.27) and promote stable ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50= 8.11-8.17) that results in a decrease in H3K27me3 levels in cells. Interestingly, EED-targeting PROTACs induce rapid degradation of EED, as well as its associated proteins, including EZH2 and SUZ12 in the PRC2 complex. Inhibition of the ubiquitin proteasome pathway abrogates PROTAC-mediated degradation of EED and its associated proteins. Furthermore, the EED targeting PROTACs selectively inhibit proliferation and survival of PRC2-dependent cancer cells (GI50= 49-58 nM). In summary, our data demonstrate a novel therapeutic modality in treating PRC2 dependent cancer through PROTAC mediated platform. Citation Format: Jessie Hao-Ru Hsu, Timothy Rasmusson, James Robinson, Fiona Pachl, Jon Read, Sameer Kawatkar, Daniel H O'Donovan, Sharan Bagal, Erin Code, Philip Rawlins, Argyrides Argyrou, Ronald Tomlinson, Ning Gao, Xiahui Zhu, Elisabetta Chiarparin, Kelly Jacques, Minhui Shen, Haley Woods, Emma Bednarski, David M. Wilson, Lisa Drew, M. Paola Castaldi, Stephen Fawell, Andrew Bloecher. EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A09. doi:10.1158/1535-7163.TARG-19-LB-A09

Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.