Search

Arginase is a promising immuno-oncology target that can restore the innate immune response. However, it's highly polar active site often requires potent inhibitors to mimic amino acids, leading to poor passive permeability and low oral exposure. Using structure-based drug design, we discovered a novel proline-based arginase inhibitor ( 10 ) that was potent but had low oral bioavailability in rat. This issue was addressed by incorporating amino acids to target PepT1/2 active transport, followed by in vivo hydrolysis post absorption. The hydrolysis rate was highly tunable, and the valine prodrug ( 19 ) showed the best balance of stability and exposure of the potent payload. Dosing of 19 in mouse xenograft models significantly increased arginine in the tumor microenvironment, resulting in tumor growth inhibition as a monotherapy and in combination with an anti-PD-L1 antibody. Compound 19 (AZD0011) displays good pharmacokinetics and was selected as a clinical drug candidate for cancer.

Arginase has long been a target of interest in immuno-oncology, but discovering an orally bioavailable inhibitor is severely constrained by the requisite boronic acid pharmacophore. We began our drug discovery campaign by building off the β-position of the literature inhibitor ABH ( 1 ). A divergent synthesis with an Ireland-Claisen rearrangement as the key step allowed access to numerous compounds, some of which we crystallized in the active site of arginase 2. We subsequently used structure-based drug design to further improve the potency of this series, ultimately achieving an inhibitor with an IC 50 value of 12 nM. Many compounds in this series were designed to behave as prodrugs, releasing their payload with up to 4-fold improved oral exposure relative to the parent. Subtle stereochemical differences between these various inhibitors and prodrugs had substantial effects on potency and pharmacokinetics.

Herein, we report the optimization of a series of epidermal growth factor receptor (EGFR) Exon20 insertion (Ex20Ins) inhibitors using structure-based drug design (SBDD), leading to the discovery of compound 28 , a potent and wild type selective molecule, which demonstrates efficacy in multiple EGFR Ex20Ins xenograft models and blood-brain barrier penetration in preclinical species. Building on our earlier discovery of an in vivo probe, SBDD was used to design a novel bicyclic core with a lower molecular weight to facilitate blood-brain barrier penetration. Further optimization including strategic linker replacement and diversification of the ring system interacting with the c-helix enabled photolytic and metabolic stability improvements. Together with refinement of molecular properties important for achieving high brain exposure, including molecular weight, H-bonding, and polarity, 28 was identified.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 ( 21 ), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

In early drug discovery, hydrolytic chemical stability is routinely assessed to ensure future developability of quality compounds and stability in in vitro test environments. When conducting high-throughput hydrolytic stability analyses as part of the compound risk assessment, aggressive conditions are typically applied to allow for faster screening. However, it can be challenging to extrapolate the real stability risk and to rank compounds due to over-estimating risk based on aggressive conditions and the narrow discriminative window. In this study, critical assay parameters including temperature, concentration, and detection technique were systematically assessed using selected model compounds, and the impact and interplay of these parameters on predictive power and prediction quality were evaluated. Improved data quality was achieved using high sample concentration and reduced temperature, combined with ultraviolet (UV) detection, while mass spectrometry (MS) detection was found to be a useful complementary detection technique. Therefore, a highly discriminative stability protocol with optimized assay parameters and experimental data quality is proposed. The optimized assay can provide early guidance on the potential stability risk of a drug molecule as well as enable more confident decision-making in compound design, selection, and development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically., (©2023 American Association for Cancer Research.)

Herein, we report a photoredox-catalyzed decarboxylative addition of N-substituted acetic acids to aldehydes to generate secondary alcohols under mild reaction conditions. Protic solvents were found to be critical to the successful implementation of this methodology. This strategy enables the formation of a novel C-C bond between aldehydes and N-substituted acetic acid derivatives of weakly nucleophilic and medicinally relevant heteroaryls such as indoles, pyrroles, indazoles, and azaindoles.

In laboratories with multiple identical analytical instruments and consistent sample workflows, analysts frequently perform repetitive software control steps, yet automation options in vendor-supplied instrument software are generally limited and may not support the desired laboratory workflow. Scripts that automate tasks to monitor systems, streamline worklist creation, or minimize downtime can save valuable personnel time and reduce errors. AutoHotkey is a free, open-source scripting language for Windows that allows users with no programming experience to easily create scripts automating a wide variety of activities. The scripts automate the tasks that a user performs while interacting with the instrument control software, such as mouse clicks and keyboard entries and closing software windows, rather than modify the underlying instrument software, and thus these scripts are compatible with multiple vendor software packages and Windows OS versions. The scripts can be triggered manually from a desktop icon or automatically through Windows Task Scheduler., (Copyright © 2021 AstraZeneca Pharmaceuticals LP. Published by Elsevier Inc. All rights reserved.)

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.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S -adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro . In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.

In this report, we describe a new photoredox catalyzed 1,4-conjugate addition of N -substituted acetic acids to electron-deficient olefins via decarboxylative C-C bond formation. This C-C bond formation occurred under mild conditions enabled by visible light irradiation. This transformation facilitated the synthesis of biologically relevant N -substituted heterocyclic structural motifs not readily accessible by other methods. The C-C bond formation protocol was applied to weakly nucleophilic heterocycles such as indoles, indazoles, imidazoles, and cyclic amides to form functionalized drug-like small molecule.

Over the last ten years, targeted covalent inhibition has become a key discipline within medicinal chemistry research, most notably in the development of oncology therapeutics. One area where this approach is underrepresented, however, is in targeting protein-protein interactions. This is primarily because these hydrophobic interfaces lack appropriately located cysteine residues to allow for standard conjugate addition chemistry. Herein, we report our development of the first covalent inhibitors of the antiapoptotic protein B-cell lymphoma extra-large (Bcl-xL), utilizing a sulfonyl fluoride (SF) warhead to selectively covalently modify tyrosine 101 of the BH3 domain-binding groove. These compounds display time-dependent inhibition in a biochemical assay and are cellularly active (U266B1). In addition, compound 7 was further elaborated to generate a chemical-biology probe molecule, which may find utility in understanding the intricacies of Bcl-xL biology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related S VI -F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

A visible-light-promoted iridium photoredox and nickel dual-catalyzed cross-coupling procedure for the formation C-N bonds has been developed. With this method, various aryl amines were chemoselectively cross-coupled with electronically and sterically diverse aryl iodides and bromides to forge the corresponding C-N bonds, which are of high interest to the pharmaceutical industries. Aryl iodides were found to be a more efficient electrophilic coupling partner. The coupling reactions were carried out at room temperature without the rigorous exclusion of molecular oxygen, thus making this newly developed Ir-photoredox/Ni dual-catalyzed procedure very mild and operationally simple., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series., (© 2014 Society for Laboratory Automation and Screening.)

We present a comprehensive study of C6-alkylidene containing oxapenems. We show that this class of β-lactamase inhibitors possesses an unprecedented spectrum with activity against class A, C, and D enzymes. Surprisingly, this class of compounds displayed significant photolytic instability in addition to the known hydrolytic instability. Quantum mechanical calculations were used to develop models to predict the stability of new analogues.