Discovery of a selective c-MET inhibitor with a novel binding mode.

The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.

Correction: Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.

A general review of the biomechanics of root anchorage.

With few exceptions, terrestrial plants are anchored to substrates by roots that experience bending and twisting forces resulting from gravity- and wind-induced forces. Mechanical failure occurs when these forces exceed the flexural or torsional tolerance limits of stems or roots, or when roots are dislodged from their substrate. The emphasis of this review is on the general principles of anchorage, how the mechanical failure of root anchorage can be averted, and recommendations for future research.

Measurement accuracy and uncertainty in plant biomechanics.

All scientific measurements are affected to some degree by both systematic and random errors. The quantification of these errors supports correct interpretation of data, thus supporting scientific progress. Absence of information regarding reliability and accuracy can slow scientific progress, and can lead to a reproducibility crisis. Here we consider both measurement theory and plant biomechanics literature. Drawing from measurement theory literature, we review techniques for assessing both the accuracy and uncertainty of a measurement process. In our survey of plant biomechanics literature, we found that direct assessment of measurement accuracy and uncertainty is not yet common. The advantages and disadvantages of efforts to quantify measurement accuracy and uncertainty are discussed. We conclude with recommended best practices for improving the scientific rigor in plant biomechanics through attention to the issues of measurement accuracy and uncertainty.

Biomechanical phenotyping pipeline for stalk lodging resistance in maize.

Stalk lodging (structural failure crops prior to harvest) significantly reduces annual yields of vital grain crops. The lack of standardized, high throughput phenotyping methods capable of quantifying biomechanical plant traits prevents comprehensive understanding of the genetic architecture of stalk lodging resistance. A phenotyping pipeline developed to enable higher throughput biomechanical measurements of plant traits related to stalk lodging is presented. The methods were developed using principles from the fields of engineering mechanics and metrology and they enable retention of plant-specific data instead of averaging data across plots as is typical in most phenotyping studies. This pipeline was specifically designed to be implemented in large experimental studies and has been used to phenotype over 40,000 maize stalks. The pipeline includes both lab- and field-based phenotyping methodologies and enables the collection of metadata. Best practices learned by implementing this pipeline over the past three years are presented. The specific instruments (including model numbers and manufacturers) that work well for these methods are presented, however comparable instruments may be used in conjunction with these methods as seen fit.•Efficient methods to measure biomechanical traits and record metadata related to stalk lodging.•Can be used in studies with large sample sizes (i.e., > 1,000).

Fragment-Based Discovery of Novel MUS81 Inhibitors.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-µM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.

Identification and Evaluation of Reversible Covalent Binders to Cys55 of Bfl-1 from a DNA-Encoded Chemical Library Screen.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

Discovery and In Vivo Efficacy of AZ-PRMT5i-1, a Novel PRMT5 Inhibitor with High MTA Cooperativity.

PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.

The semi-automated development of plant cell wall finite element models.

This study presents a methodology for a high-throughput digitization and quantification process of plant cell walls characterization, including the automated development of two-dimensional finite element models. Custom algorithms based on machine learning can also analyze the cellular microstructure for phenotypes such as cell size, cell wall curvature, and cell wall orientation. To demonstrate the utility of these models, a series of compound microscope images of both herbaceous and woody representatives were observed and processed. In addition, parametric analyses were performed on the resulting finite element models. Sensitivity analyses of the structural stiffness of the resulting tissue based on the cell wall elastic modulus and the cell wall thickness; demonstrated that the cell wall thickness has a three-fold larger impact of tissue stiffness than cell wall elastic modulus.

Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function.

Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nε-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nε-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

High-throughput interrogation of ligand binding mode using a fluorescence-based assay.

Probing the pocket: A high-throughput fluorescence-based thermal shift (FTS) assay utilized different forms of a protein (in gray) to establish the binding mode of a ligand (see picture). The assay serves in the rapid evaluation of structure-activity binding-mode relationships for a series of ligands of Plk1, an important target of anticancer therapy.

Maize stalk stiffness and strength are primarily determined by morphological factors.

The maize (Zea mays) stem is a biological structure that must balance both biotic and structural load bearing duties. These competing requirements are particularly relevant in the design of new bioenergy crops. Although increased stem digestibility is typically associated with a lower structural strength and higher propensity for lodging, with the right balance between structural and biological activities it may be possible to design crops that are high-yielding and have digestible biomass. This study investigates the hypothesis that geometric factors are much more influential in determining structural strength than tissue properties. To study these influences, both physical and in silico experiments were used. First, maize stems were tested in three-point bending. Specimen-specific finite element models were created based on x-ray computed tomography scans. Models were validated by comparison with experimental data. Sensitivity analyses were used to assess the influence of structural parameters such as geometric and material properties. As hypothesized, geometry was found to have a much stronger influence on structural stability than material properties. This information reinforces the notion that deficiencies in tissue strength could be offset by manipulation of stalk morphology, thus allowing the creation of stalks which are both resilient and digestible.

High throughput phenotyping of cross-sectional morphology to assess stalk lodging resistance.

BACKGROUND: Stalk lodging (mechanical failure of plant stems during windstorms) leads to global yield losses in cereal crops estimated to range from 5% to 25% annually. The cross-sectional morphology of plant stalks is a key determinant of stalk lodging resistance. However, previously developed techniques for quantifying cross-sectional morphology of plant stalks are relatively low-throughput, expensive and often require specialized equipment and expertise. There is need for a simple and cost-effective technique to quantify plant traits related to stalk lodging resistance in a high-throughput manner. RESULTS: A new phenotyping methodology was developed and applied to a range of plant samples including, maize (Zea mays), sorghum (Sorghum bicolor), wheat (Triticum aestivum), poison hemlock (Conium maculatum), and Arabidopsis (Arabis thaliana). The major diameter, minor diameter, rind thickness and number of vascular bundles were quantified for each of these plant types. Linear correlation analyses demonstrated strong agreement between the newly developed method and more time-consuming manual techniques (R2 > 0.9). In addition, the new method was used to generate several specimen-specific finite element models of plant stalks. All the models compiled without issue and were successfully imported into finite element software for analysis. All the models demonstrated reasonable and stable solutions when subjected to realistic applied loads. CONCLUSIONS: A rapid, low-cost, and user-friendly phenotyping methodology was developed to quantify two-dimensional plant cross-sections. The methodology offers reduced sample preparation time and cost as compared to previously developed techniques. The new methodology employs a stereoscope and a semi-automated image processing algorithm. The algorithm can be used to produce specimen-specific, dimensionally accurate computational models (including finite element models) of plant stalks.

Biomimicking interfacial fracture behavior of lizard tail autotomy with soft microinterlocking structures.

Biological soft interfaces often exhibit complex microscale interlocking geometries to ensure sturdy and flexible connections. If needed, the interlocking can rapidly be released on demand leading to an abrupt decrease of interfacial adhesion. Here, inspired by lizard tail autotomy where such apparently tunable interfacial fracture behavior can be observed, we hypothesized an interlocking mechanism between the tail and body based on the muscle-actuated mushroom-shaped microinterlocks along the fracture planes. To mimic the fracture behavior of the lizard tail, we developed a soft bilayer patch that consisted of a dense array of soft hemispherical microstructures in the upper layer acting as mechanical interlocks with the counter body part. The bottom control layer contained a microchannel that allowed to deflect the upper layer when applying the negative pressure, thus mimicking muscle contraction. In the microinterlocked condition, the biomimetic tail demonstrated a 2.7-fold and a three-fold increase in adhesion strength and toughness, respectively, compared to the pneumatically released microinterlocks. Furthermore, as per the computational analysis, the subsurface microchannel in the control layer enabled augmented adhesion by rendering the interface more compliant as a dissipative matrix, decreasing contact opening and strain energy dissipation by 50%. The contrasting features between the microinterlocked and released cases demonstrated a highly tunable adhesion of our biomimetic soft patch. The potential applications of our study are expected in soft robotics and prosthetics.

Biomimetic fracture model of lizard tail autotomy.

Lizard tail autotomy is an antipredator strategy consisting of sturdy attachment at regular times but quick detachment during need. We propose a biomimetic fracture model of lizard tail autotomy using multiscale hierarchical structures. The structures consist of uniformly distributed micropillars with nanoporous tops, which recapitulate the high-density mushroom-shaped microstructures found on the lizard tail's muscle fracture plane. The biomimetic experiments showed adhesion enhancement when combining nanoporous interfacial surfaces with flexible micropillars in tensile and peel modes. The fracture modeling identified micro- and nanostructure-based toughening mechanisms as the critical factor. Under wet conditions, capillarity-assisted energy dissipation pertaining to liquid-filled microgaps and nanopores further increased the adhesion performance. This research presents insights on lizard tail autotomy and provides new biomimetic ideas to solve adhesion problems.

Cross-sectional geometry predicts failure location in maize stalks.

BACKGROUND: Stalk lodging (breaking of agricultural plant stalks prior to harvest) is a multi-billion dollar a year problem. Stalk lodging occurs when high winds induce bending moments in the stalk which exceed the bending strength of the plant. Previous biomechanical models of plant stalks have investigated the effect of cross-sectional morphology on stalk lodging resistance (e.g., diameter and rind thickness). However, it is unclear if the location of stalk failure along the length of stem is determined by morphological or compositional factors. It is also unclear if the crops are structurally optimized, i.e., if the plants allocate structural biomass to create uniform and minimal bending stresses in the plant tissues. The purpose of this paper is twofold: (1) to investigate the relationship between bending stress and failure location of maize stalks, and (2) to investigate the potential of phenotyping for internode-level bending stresses to assess lodging resistance. RESULTS: 868 maize specimens representing 16 maize hybrids were successfully tested in bending to failure. Internode morphology was measured, and bending stresses were calculated. It was found that bending stress is highly and positively associated with failure location. A user-friendly computational tool is presented to help plant breeders in phenotyping for internode-level bending stress. Phenotyping for internode-level bending stresses could potentially be used to breed for more biomechanically optimal stalks that are resistant to stalk lodging. CONCLUSIONS: Internode-level bending stress plays a potentially critical role in the structural integrity of plant stems. Equations and tools provided herein enable researchers to account for this phenotype, which has the potential to increase the bending strength of plants without increasing overall structural biomass.