Site-specific Quadruple-Functionalised Antibodies.

Antibody-drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi-payload ADCs, have been developed. Here we deploy multiple orthogonal site-specific protein modification strategies to generate highly homogeneous multi-functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site-specific cyclopropenone (CPO)-based reagent, represents the first example of a homogeneous multi-payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs.

Identification and optimization of a novel series of selective PIP5K inhibitors.

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

Vinylpyridine as a Tunable Covalent Warhead Targeting C797 in EGFR.

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.

Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

Chemical Biology Approaches Confirm MCT4 as the Therapeutic Target of a Cellular Optimized Hit.

Lactic acid transport is a key process maintaining glycolytic flux in tumors. Inhibition of this process will result in glycolytic shutdown, impacting on cell growth and survival and thus has been pursued as a therapeutic approach for cancers. Using a cell-based screen in a MCT4-dependent cell line, we identified and optimized compounds for their ability to inhibit the efflux of intracellular lactic acid with good physical and pharmacokinetic properties. To deconvolute the mechanism of lactic acid efflux inhibition, we have developed three assays to measure cellular target engagement. Specifically, we synthesized a biologically active photoaffinity probe (IC50 < 10 nM), and using this probe, we demonstrated selective engagement of MCT4 of our parent molecule through a combination of confocal microscopy and in-cell chemoproteomics. As an orthogonal assay, the cellular thermal shift assay (CETSA) confirmed binding to MCT4 in the cellular system. Comparisons of lactic acid efflux potencies in cells with differential expression of MCT family members further confirmed that the optimized compounds inhibit the efflux of lactic acid through the inhibition of MCT4. Taken together, these data demonstrate the power of orthogonal chemical biology methods to determine cellular target engagement, particularly for proteins not readily amenable to traditional biophysical methods.

Discovery of the Potent and Selective Inhaled Janus Kinase 1 Inhibitor AZD4604 and Its Preclinical Characterization.

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

Discovery of Clinical Candidate AZD0095, a Selective Inhibitor of Monocarboxylate Transporter 4 (MCT4) for Oncology.

Due to increased reliance on glycolysis, which produces lactate, monocarboxylate transporters (MCTs) are often upregulated in cancer. MCT4 is associated with the export of lactic acid from cancer cells under hypoxia, so inhibition of MCT4 may lead to cytotoxic levels of intracellular lactate. In addition, tumor-derived lactate is known to be immunosuppressive, so MCT4 inhibition may be of interest for immuno-oncology. At the outset, no potent and selective MCT4 inhibitors had been reported, but a screen identified a triazolopyrimidine hit, with no close structural analogues. Minor modifications to the triazolopyrimidine were made, alongside design of a constrained linker and broad SAR exploration of the biaryl tail to improve potency, physical properties, PK, and hERG. The resulting clinical candidate 15 (AZD0095) has excellent potency (1.3 nM), MCT1 selectivity (>1000×), secondary pharmacology, clean mechanism of action, suitable properties for oral administration in the clinic, and good preclinical efficacy in combination with cediranib.

Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

AZD4625 is a Potent and Selective Inhibitor of KRASG12C.

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line-derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents.

Discovery of AZD4625, a Covalent Allosteric Inhibitor of the Mutant GTPase KRASG12C.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Inhibitors of the tyrosine kinase EphB4. Part 4: Discovery and optimization of a benzylic alcohol series.

Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good kinase selectivity profile, good physical properties and pharmacokinetic parameters, suggesting it is a suitable candidate to investigate the therapeutic potential of EphB4 kinase inhibitors.

Drugging the undruggable: a computational chemist's view of KRASG12C.

In recent years, the emergence of targeted covalent inhibitors which bind to the G12C mutant of KRAS have offered a solution to this previously intractable target. Inhibitors of KRASG12C tend to be structurally complex, displaying features such as atropisomerism, chiral centres and a reactive covalent warhead. Such molecules result in lengthy and challenging syntheses, and as a consequence critical decisions need to be made at the design level to maximise the chances of success. Here we take a retrospective look into how computational chemistry can help guide and prioritise medicinal chemistry efforts in the context of a series of conformationally restricted tetracyclic quinolines.

Toward the comprehensive systematic enumeration and synthesis of novel kinase inhibitors based on a 4-anilinoquinazoline binding mode.

There are currently eight small-molecule kinase inhibitors approved as cancer treatments, and a significantly larger number of compounds are in the earlier stages of clinical development. Although kinase inhibitors are most commonly developed in a cancer setting, other disease areas have been targeted. The vast majority of reported kinase small-molecule inhibitors contain functionalities that interact with the adenosine triphosphate (ATP) binding site of the kinase. The 4-anilinoquinazolines have previously been reported as potent epidermal growth factor receptor (EGFR) inhibitors, binding at the 'hinge' region of the ATP site. Subsequently, this chemical series has been optimized against a number of different kinases including Src and Aurora B. Here, we detail the computational enumeration of ring systems that have the ability to make comparable interactions to the 4-anilinoquinazoline core. These were prioritized by computational, medicinal, and synthetic chemistry input, and a number of libraries were subsequently synthesized.

Covalent inhibitors of the GTPase KRASG12C: a review of the patent literature.

Introduction: KRAS is one of the most important oncology drug targets, playing a pivotal role in the initiation and progression of many human tumors. It has long been held undruggable due to many previously failed attempts to both directly and indirectly target this challenging GTPase protein family.Areas covered: This review covers patent applications claiming inhibitors of the mutant GTPase KRASG12C that act via covalent modification of cysteine at codon 12 in the period of 2014 to the present. A total of 37 PCT applications from 9 applicants are evaluated, with the discussion organized alphabetically by assignee name.Expert opinion: The last 5 years have seen an explosion in interest around this important target with many companies aiming to capitalize on the breakthrough discovery of covalent allosteric inhibitors of the glycine to cysteine mutant form of the enzyme. The first agents from this effort have now entered clinical trials and preliminary data are encouraging with responses seen in both lung adenocarcinoma and colorectal cancer patients.

The Pharmacokinetic-Pharmacodynamic (PKPD) Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range of Mouse Xenograft Models of GIST.

PURPOSE: The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic-pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors. EXPERIMENTAL DESIGN: A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines. RESULTS: AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations. CONCLUSIONS: We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.

Discovery and pharmacological characterization of AZD3229, a potent KIT/PDGFRα inhibitor for treatment of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

Discovery of (2R)-N-[3-[2-[(3-Methoxy-1-methyl-pyrazol-4-yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide (AZD4205) as a Potent and Selective Janus Kinase 1 Inhibitor.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

Structure-Based Design and Pharmacokinetic Optimization of Covalent Allosteric Inhibitors of the Mutant GTPase KRASG12C.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.