Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex.

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4, and mediate potent PTPN2/N1 degradation in cells and mice.

Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination.

The CryoEM structure of human serum albumin in complex with ligands.

Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution of endobiotics and xenobiotics within the organism. In this paper we report the cryoEM structures of HSA in the apo form and in complex with two ligands (salicylic acid and teniposide) at a resolution of 3.

High-Resolution Cryo-Electron Microscopy Structure Determination of Tellurite-Resistance Protein A via 200 kV Transmission Electron Microscopy.

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins' native conformations, thus affecting stability and functionality.

Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A.

Structure Guided Discovery of Novel Pan Metallo-β-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of β-lactam (BL) and β-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-β-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by , discovered earlier as metallo-beta-lactamase inhibitors, was selected for virtual screening.

Absolute configuration determination of pharmaceutical crystalline powders by MicroED chiral salt formation.

Microcrystal electron diffraction (MicroED) has established its complementary role alongside X-ray diffraction in crystal structure elucidation. Unfortunately, kinematical refinement of MicroED data lacks the differentiation power to assign the absolute structure solely based on the measured intensities. Here we report a method for absolute configuration determination MicroED by employing salt formation with chiral counterions.

Functionally selective signaling and broad metabolic benefits by novel insulin receptor partial agonists.

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g.

Discovery of : an Efficient Inhibitor of the HDM2-p53 Protein-Protein Interaction.

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties.

Small Molecule Microcrystal Electron Diffraction for the Pharmaceutical Industry-Lessons Learned From Examining Over Fifty Samples.

The emerging field of microcrystal electron diffraction (MicroED) is of great interest to industrial researchers working in the drug discovery and drug development space. The promise of being able to routinely solve high-resolution crystal structures without the need to grow large crystals is very appealing. Despite MicroED's exciting potential, adoption across the pharmaceutical industry has been slow, primarily owing to a lack of access to specialized equipment and expertise.

Cryo-EM structures of inhibitory antibodies complexed with arginase 1 provide insight into mechanism of action.

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size.

Pembrolizumab microgravity crystallization experimentation.

Crystallization processes have been widely used in the pharmaceutical industry for the manufacture, storage, and delivery of small-molecule and small protein therapeutics. However, the identification of crystallization processes for biologics, particularly monoclonal antibodies, has been prohibitive due to the size and the flexibility of their overall structure. There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics.

Serendipitous discovery of aryl boronic acids as β-lactamase inhibitors.

High throughput screening for β-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).

Cryo-EM for Small Molecules Discovery, Design, Understanding, and Application.

We present a perspective of our view of the application of cryoelectron microscopy (cryo-EM) to structure-based drug design (SBDD). We discuss the basic needs and requirements for SBDD, the current state of cryo-EM, and the challenges that need to be overcome for this technique to reach its full potential in facilitating the process of drug discovery.

Routine single particle CryoEM sample and grid characterization by tomography.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface.

Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis.

The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice.

Aims: Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity.

Methods: We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase.

To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives.

A novel storage system for cryoEM samples.

We present here a new CryoEM grid boxes storage system designed to simplify sample labeling, tracking and retrieval. The system is based on the crystal pucks widely used by the X-ray crystallographic community for storage and shipping of crystals. This system is suitable for any cryoEM laboratory, but especially for large facilities that will need accurate tracking of large numbers of samples coming from different sources.