Micelle-Promoted Reductive Amination of DNA-Conjugated Amines for DNA-Encoded Library Synthesis.

DNA-encoded libraries (DELs) have become a leading technology for hit identification in drug discovery projects as large, diverse libraries can be generated. DELs are commonly synthesised via split-and-pool methodology; thus, chemical transformations utilised must be highly efficient, proceeding with high conversions. Reactions performed in DEL synthesis also require a broad substrate scope to produce diverse, drug-like libraries. Many pharmaceutical compounds incorporate multiple C-N bonds, over a quarter of which are synthesised via reductive aminations. However, few on-DNA reductive amination procedures have been developed. Herein is reported the application of the micelle-forming surfactant, TPGS-750-M, to the on-DNA reductive amination of DNA-conjugated amines, yielding highly efficient conversions with a broad range of aldehydes, including medicinally relevant heterocyclic and aliphatic substrates. The procedure is compatible with DNA amplification and sequencing, demonstrating its applicability to DEL synthesis.

Lipid Tales: Optimizing Arylomycin Membrane Anchors.

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria.

Fragment expansion with NUDELs - poised DNA-encoded libraries.

Optimisation of the affinity of lead compounds is a critical challenge in the identification of drug candidates and chemical probes and is a process that takes many years. Fragment-based drug discovery has become established as one of the methods of choice for drug discovery starting with small, low affinity compounds. Due to their low affinity, the evolution of fragments to desirable levels of affinity is often a key challenge. The accepted best method for increasing the potency of fragments is by iterative fragment growing, which can be very time consuming and complex. Here, we introduce a paradigm for fragment hit optimisation using poised DNA-encoded chemical libraries (DELs). The synthesis of a poised DEL, a partially constructed library that retains a reactive handle, allows the coupling of any active fragment for a specific target protein, allowing rapid discovery of potent ligands. This is illustrated for bromodomain-containing protein 4 (BRD4), in which a weakly binding fragment was coupled to a 42-member poised DEL via Suzuki-Miyaura cross coupling resulting in the identification of an inhibitor with 51 nM affinity in a single step, representing an increase in potency of several orders of magnitude from an original fragment. The potency of the compound was shown to arise from the synergistic combination of substructures, which would have been very difficult to discover by any other method and was rationalised by X-ray crystallography. The compound showed attractive lead-like properties suitable for further optimisation and demonstrated BRD4-dependent cellular pharmacology. This work demonstrates the power of poised DELs to rapidly optimise fragments, representing an attractive generic approach to drug discovery.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

Filling a nick in NIK: Extending the half-life of a NIK inhibitor through structure-based drug design.

Inhibition of NF-κB inducing kinase (NIK) has been pursued as a promising therapeutic target for autoimmune disorders due to its highly regulated role in key steps of the NF-κB signaling pathway. Previously reported NIK inhibitors from our group were shown to be potent, selective, and efficacious, but had higher human dose projections than desirable for immunology indications. Herein we report the clearance-driven optimization of a NIK inhibitor guided by metabolite identification studies and structure-based drug design. This led to the identification of an azabicyclo[3.1.0]hexanone motif that attenuated in vitro and in vivo clearance while maintaining NIK potency and increasing selectivity over other kinases, resulting in a greater than ten-fold reduction in predicted human dose.

Trust Your Gut: Strategies and Tactics for Intestinally Restricted Drugs.

Non-absorbable small-molecule drugs targeted to the gut represent an alternative approach to safe, non-systemic therapeutics. Such drugs remain confined to the gastrointestinal tract upon oral dosing by virtue of their limited passive permeability, increasing the local concentration at the site of action while minimizing exposure elsewhere in the body. Herein we review the latest advances in the field of gut-restricted therapeutics, highlighting the different strategies and tactics that medicinal chemists have employed in pursuit of drugs with minimal intestinal absorption.

Chemical space docking enables large-scale structure-based virtual screening to discover ROCK1 kinase inhibitors.

With the ever-increasing number of synthesis-on-demand compounds for drug lead discovery, there is a great need for efficient search technologies. We present the successful application of a virtual screening method that combines two advances: (1) it avoids full library enumeration (2) products are evaluated by molecular docking, leveraging protein structural information. Crucially, these advances enable a structure-based technique that can efficiently explore libraries with billions of molecules and beyond. We apply this method to identify inhibitors of ROCK1 from almost one billion commercially available compounds. Out of 69 purchased compounds, 27 (39%) have Ki values < 10 µM. X-ray structures of two leads confirm their docked poses. This approach to docking scales roughly with the number of reagents that span a chemical space and is therefore multiple orders of magnitude faster than traditional docking.

Turning the Other Cheek: Influence of the cis-Tetrafluorocyclohexyl Motif on Physicochemical and Metabolic Properties.

The targeted introduction of substituents in order to tailor a molecule's pharmacologic, physicochemical, and metabolic properties has long been of interest to medicinal chemists. The all-cis tetrafluorocyclohexyl motif-dubbed Janus face, due to its electrostatically polarized cyclohexyl ring-represents one such example where chemists might incorporate a metabolically stable, polar, lipocompatible motif. To better understand its potential utility, we have synthesized three series of matched molecular pairs (MMPs) where each MMP differs only in the cyclohexane unit, i.e., with a tetrafluorocyclohexyl or a standard cyclohexyl motif. With the introduction of the facially polarized all-cis tetrafluorocyclohexyl ring, the resulting compounds have significantly modified physicochemical properties (e.g., kinetic solubility, lipophilicity and permeability) and metabolic stabilities. These results further speak to the promise of this substituent as a tactic to improve the drug-like properties of molecules.

When Cofactors Aren't X Factors: Functional Groups That Are Labile in Human Liver Microsomes in the Absence of NADPH.

The metabolic stability of compounds is often assessed at an early stage in drug discovery programs by profiling with hepatic microsomes. Exclusion of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in these assays provides insight into non-cytochrome P450 (CYP)-mediated metabolism. This report uses a matched molecular pair (MMP) application to assess which chemical substituents are commonly susceptible to non-NADPH-mediated metabolism by microsomes. The analysis found the overall prevalence of metabolism in the absence of NADPH to be low, with esters, amides, aldehydes, and oxetanes being among the most commonly susceptible functional groups. Given that non-CYP enzymes, such as esterases, may be expressed extrahepatically and lead to lower confidence in predicted pharmacokinetic profiles, an awareness of the functional groups that commonly undergo non-NADPH-mediated metabolism-as well as options for their replacement based on experimental MMP data-may help researchers derisk metabolic stability issues at an earlier stage in drug discovery.

Highly efficient on-DNA amide couplings promoted by micelle forming surfactants for the synthesis of DNA encoded libraries.

DNA encoded libraries (DELs) represent powerful new technology for finding small molecule ligands for proteins and are increasingly being applied to hit finding in medicinal chemistry. Crucial to the synthesis of high quality DELs is the identification of chemical reactions for their assembly that proceed with very high conversion across a range of different substrates, under conditions compatible with DNA-tagged substrates. Many current chemistries used in DEL synthesis do not meet this requirement, resulting in libraries of low fidelity. Amide couplings are the most commonly used reaction in synthesis of screening libraries and also in DELs. The ability to carry out highly efficient, widely applicable amide couplings in DEL synthesis would therefore be highly desirable. We report a method for amide coupling using micelle forming surfactants, promoted by a modified linker, that is broadly applicable across a wide range of substrates. Most significantly, this works exceptionally well for coupling of DNA-conjugated carboxylic acids (N-to-C) with amines in solution, a procedure that is currently very inefficient. The optimisation of separate procedures for coupling of DNA-conjugated acids and amines by reagent screening and statistically driven optimisation is described. The generality of the method is illustrated by the application to a wide range of examples with unprecedented levels of conversion. The utility of the (N-to-C) coupling of DNA-conjugated acids in DEL synthesis is illustrated by the three cycle synthesis of a fully DNA-encoded compound by two cycles of coupling of an aminoester, with intermediate ester hydrolysis, followed by capping with an amine. This methodology will be of great utility in the synthesis of high fidelity DELs.

Stereochemical Differences in Fluorocyclopropyl Amides Enable Tuning of Btk Inhibition and Off-Target Activity.

Bruton's tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retained-and in some cases improved-a safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.

Strategies to Mitigate the Bioactivation of Aryl Amines.

The bioactivation of xenobiotics to yield reactive metabolites can lead to tolerability and toxicity concerns within a drug discovery program. Development of strategies for mitigating the metabolic liability of commonly encountered toxicophores, such as anilines, relies on an understanding of the relative tendency of these functionalities to undergo bioactivation. In this report, we present the first systematic study of the structure-activity relationships of the bioactivation of aryl amine fragments (molecular weight < 250 Da) using a glutathione (GSH) trapping assay in the presence of human liver microsomes and the reduced form of nicotinamide adenine dinucleotide phosphate. This study demonstrates that conversion of anilines to nitrogen-containing heteroarylamines results in a lower abundance of GSH conjugates in the order phenyl > pyrimidine ≈ pyridine > pyridazine. Introduction of electron-withdrawing functionality on the aromatic ring had a less pronounced effect on the extent of GSH conjugation. Examination of more drug-like compounds sourced from in-house drug discovery programs revealed similar trends in bioactivation between matched pairs containing (hetero)aryl amines. This study provides medicinal chemists with insights and qualitative guidance for the minimization of risks related to aryl amine metabolism.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

LogD Contributions of Substituents Commonly Used in Medicinal Chemistry.

The importance of physicochemical property calculation and measurement is well-established in drug discovery. In particular, lipophilicity predictions play a central role in target design and prioritization. While significant progress has been made in our ability to calculate both logP and logD, the quality of these predictions is limited by the size and diversity of the underlying data set. Access to diverse data sets and advanced models is often limited to large organizations or consortia, and they are not available to many students and practitioners of medicinal chemistry. A molecular matched pair analysis of median ΔlogD 7.4 contributions for substituents commonly used in drug discovery programs at Genentech is reported. The results of this ΔlogD analysis are compiled into a single table, which we anticipate will be of use to practicing medicinal chemists.

Safety Considerations in the Development of Hippo Pathway Inhibitors in Cancers.

The Hippo pathway is a critical regulator of cell and organ growth and has emerged as a target for therapeutic intervention in cancers. Its signaling is thought to play an important role in various physiological processes including homeostasis and tissue regeneration. To date there has been limited information about potential pharmacology-related (on-target) safety liabilities of Hippo pathway inhibitors in the context of cancer indications. Herein, we review data from human genetic disorders and genetically engineered rodent models to gain insight into safety liabilities that may emerge from the inhibition of Hippo pathway. Germline systemic deletion of murine Hippo pathway effectors (Yap, Taz, and Teads) resulted in embryonic lethality or developmental phenotypes. Mouse models with tissue-specific deletion (or mutant overexpression) of the key effectors in Hippo pathways have indicated that, at least in some tissues, Hippo signaling may be dispensable for physiological homeostasis; and appears to be critical for regeneration upon tissue damage, indicating that patients with underlying comorbidities and/or insults caused by therapeutic agents and/or comedications may have a higher risk. Caution should be taken in interpreting phenotypes from tissue-specific transgenic animal models since some tissue-specific promoters are turned on during development. In addition, therapeutic agents may result in systemic effects not well-predicted by animal models with tissue-specific gene deletion. Therefore, the development of models that allows for systemic deletion of Yap and/or Taz in adult animals will be key in evaluating the potential safety liabilities of Hippo pathway modulation. In this review, we focus on potential challenges and strategies for targeting the Hippo pathway in cancers.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Hippo Pathway in Cancer: Aberrant Regulation and Therapeutic Opportunities.

The Hippo pathway remains a central focus in both basic and translational research and is a key modulator of developmental biology. Dysregulation of the pathway is associated with a plethora of human cancers and there are multiple efforts to target key components of the pathway for disease intervention. In this review, we briefly highlight the latest research advances around the core components of the Hippo pathway in cancer. More specifically, we discuss several genetic aberrations of these factors as mechanisms for the development of cancers, including genetic amplification, deletion, and gene fusions. Additionally, we highlight the role of the Hippo pathway in cancer therapy resistance and tumor immunogenicity. Last, we summarize the ongoing efforts to target the pathway in cancers.

An Integrated Analysis of Solid Form Change Impact on Solubility and Permeability: Case Study of Oral Exposure in Rats of an RAR Related Orphan Receptor C Inhibitor.

It is well acknowledged that the oral absorption of a drug can be influenced by its solubility, which is usually associated with its solid form properties. G1032 is a retinoic acid-related orphan receptor inverse agonist. Crystalline solid (form A) was identified with an aqueous solubility of 130 µg/mL. This form was used in an oral dose escalation study in rodents up to 300 mg/kg and achieved good exposures. Later on, a more stable crystalline hydrate (form B) was identified and the aqueous solubility was reduced to 55 µg/mL. A modeling exercise suggested that this solubility change would cause a 2-fold decrease in exposure at tested doses; however, the actual reduction was far larger than the model predicted. At high dose, exposure was found to be reduced by almost 10-fold. A parameter sensitivity analysis suggested that such a drop in exposure could be associated with permeability reduction as well. More in vitro permeability experiments were performed, indicating G1032 was an efflux transporter substrate. This finding was integrated into the modeling and the design for in vivo studies. Data obtained from those studies allowed us to better understand the causes of the higher-than-expected exposure change and enabled decision-making.

Hippo pathway inhibition by blocking the YAP/TAZ-TEAD interface: a patent review.

Introduction: The Hippo pathway represents a new and intriguing opportunity for the treatment of cancer. Activation or overexpression of Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ) has been shown to lead to cell transformation and tumor development. To date, no small molecule compounds targeting this pathway have progressed to the clinic, illustrating both its potential and its infancy. Areas covered: The present review seeks to summarize published patent applications from assignee companies that have disclosed direct small molecule inhibitors of the YAP/TAZ-transcriptional enhanced associate domain (TEAD) interaction. Expert opinion: The Hippo pathway, and specifically the YAP/TAZ-TEAD transcriptional complex, has been shown to be a promising target for the treatment of cancer. However, reports in the area of small molecules targeting the YAP/TAZ-TEAD transcriptional activation complex are few and far between, with only two published patent applications that disclose compounds with moderate levels of pathway inhibition. Interestingly, the YAP/TAZ-TEAD complex can be disrupted through two very different mechanisms, one of which is direct inhibition at either the Ω-loop or the α-helix of the YAP-TEAD binding interface. Both YAP protein segments have been shown to be important to TEAD binding. Alternatively, it has been reported that allosteric inhibition might be accomplished by binding the TEAD palmitoylation pocket, thus disrupting YAP binding and also native protein stabilization. The advantages and liabilities of disrupting the YAP/TAZ-TEAD complex through these two distinct mechanisms have yet to be fully elucidated, and it remains unclear which approach, if any, will generate the first clinical stage inhibitor of the Hippo pathway.