High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

Activation of Hepatocyte Growth Factor/MET Signaling as a Mechanism of Acquired Resistance to a Novel YAP1/TEAD Small Molecule Inhibitor.

Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of cases are considered YAP1/TEAD dependent. Identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs, which may be necessary for YAP1 protein interactions, has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TEAD complex formation and transcriptional activity. We report the discovery and characterization of a novel YAP1/TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TEAD activity. In vivo, MRK-A showed a favorable tolerability profile in mice and demonstrated pharmacokinetics suitable for twice daily oral dosing in preclinical efficacy studies. Importantly, monotherapeutic targeting of YAP1/TEAD in preclinical models generated regressions in a mesothelioma CDX model; however, rapid resistance to therapy was observed. RNA-sequencing of resistant tumors revealed mRNA expression changes correlated with the resistance state and a marked increase of hepatocyte growth factor (HGF) expression. In vitro, exogenous HGF was able to fully rescue cytostasis induced by MRK-A in mesothelioma cell lines. In addition, co-administration of small molecule inhibitors of the MET receptor tyrosine kinase suppressed the resistance generating effect of HGF on MRK-A induced growth inhibition. In this work, we report the structure and characterization of MRK-A, demonstrating potent and specific inhibition of YAP1/TAZ-TEAD-mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling, including factors resulting in acquired drug resistance.

Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.

Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.

Design and synthesis of unprecedented 9- and 10-membered cyclonucleosides with PRMT5 inhibitory activity.

Synthesis of medium-sized rings is known to be challenging due to high transannular strain especially for 9- and 10-membered rings. Herein we report design and synthesis of unprecedented 9- and 10-membered purine 8,5'-cyclonucleosides as the first cyclonucleoside PRMT5 inhibitors. The cocrystal structure of PRMT5:MEP50 in complex with the synthesized 9-membered cyclonucleoside 1 revealed its binding mode in the SAM binding pocket of PRMT5.

Discovery and characterization of novel peptide inhibitors of the NRF2/MAFG/DNA ternary complex for the treatment of cancer.

Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).

The Discovery of Two Novel Classes of 5,5-Bicyclic Nucleoside-Derived PRMT5 Inhibitors for the Treatment of Cancer.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.

Allosteric Modulation of Protein Arginine Methyltransferase 5 (PRMT5).

Protein arginine methyltransferase 5 (PRMT5) belongs to a family of enzymes that regulate the posttranslational modification of histones and other proteins via methylation of arginine. Methylation of histones is linked to an increase in transcription and regulates a manifold of functions such as signal transduction and transcriptional regulation. PRMT5 has been shown to be upregulated in the tumor environment of several cancer types, and the inhibition of PRMT5 activity was identified as a potential way to reduce tumor growth. Previously, four different modes of PRMT5 inhibition were known-competing (covalently or non-covalently) with the essential cofactor S-adenosyl methionine (SAM), blocking the substrate binding pocket, or blocking both simultaneously. Herein we describe an unprecedented conformation of PRMT5 in which the formation of an allosteric binding pocket abrogates the enzyme's canonical binding site and present the discovery of potent small molecule allosteric PRMT5 inhibitors.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism.

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis.

8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.

Discovery of novel BTK inhibitors with carboxylic acids.

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions.

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers.

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

Discovery of 8-Amino-imidazo[1,5-a]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.

Dissecting Therapeutic Resistance to ERK Inhibition.

The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V(600E)) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAF(V600) (E)-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models. ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G(186D)) in the DFG motif of ERK1. Structural and biophysical studies demonstrated specific defects in SCH772984 binding to mutant ERK. Taken together, these studies describe the interaction of SCH772984 with ERK and identify a novel mechanism of ERK inhibitor resistance through mutation of a single residue within the DFG motif. Mol Cancer Ther; 15(4); 548-59. ©2016 AACR.

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.