The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.

We report bio-structural, bio-chemical and bio-physical evidence demonstrating how small molecules can bind to both wild-type and mutant IDH1, but only inhibit the enzymatic activity of the mutant isoform. Enabled through x-ray crystallography, we characterized a series of small molecule inhibitors that bound to mutant IDH1 differently than the marketed inhibitor Ivosidenib, for which we have determined the x-ray crystal structure. Across the industry several mutant IDH1 inhibitor chemotypes bind to this allosteric IDH1 pocket and selectively inhibit the mutant enzyme. Detailed characterization by a variety of biophysical techniques and NMR studies led us to propose how compounds binding in the allosteric IDH1 R132H pocket inhibit the production of 2-Hydroxy glutarate.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.

TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Abstract Background Respiratory syncytial virus (RSV) infection is a major public health burden for infants and the elderly worldwide. Currently, there are no approved vaccines and only one moderately effective marketed antibody (Synagis®) for the prevention of RSV infection in high-risk infants. Sampling the human antibody repertoire has led to the realization that the RSV fusion (F) protein in its prefusion conformation is the preferred target for potent neutralizing antibodies and thus makes the protein an attractive candidate for vaccine developers. Methods We report the isolation of a potent and broad RSV neutralizing monoclonal antibody (mAb), which was discovered through molecular cloning of cultured RSV postfusion F protein-baited single-sorted human memory B cells. The epitope for the mAb was mapped to RSV F protein using various methods; including X-ray crystallography, alanine scan, and RSV escape mutant generation Results The mAb binds to both RSV pre- and postfusion F proteins at site IV and can neutralize RSV A and B laboratory strains with subnanomolar potency, superior to that of Palivizumab. Antigenic site IV is conserved between pre- and postfusion F proteins in both RSV A and B subgroups, and sequence alignment showed that the mAb-binding site was conserved in >1,000 RSV A and B clinical isolates. In vivo cotton rat studies demonstrated protection of both the upper and lower respiratory tract of antibody-infused animals challenged with either RSV A or RSV B. Conclusion Overall, the fully human mAb we have isolated has great potential to be developed for passive immune-prophylaxis in infants. A prevalent view of the RSV scientific community is that RSV neutralizing mAbs in human sera primarily target the prefusion F protein and predominantly bind antigenic site Ø. In contrast, our finding demonstrates that very broad and potent RSV neutralizing mAb can also recognize sites common to pre- and postfusion F proteins. Furthermore, the RSV F antigenic site IV presents a neutralizing epitope which is highly conserved. Therefore, it is worthwhile to consider site IV, in addition to site Ø, in the design of RSV subunit vaccines. Disclosures K. Vora, Merck: Employee, Salary. Z. Chen, Merck: Employee, Salary. H. P. Su, Merck: Employee, Salary. A. Tang, Merck: Employee, Salary. K. Cox, merck: Employee, Salary. C. Callahan, Merck: Employee, Salary. L. Zhang, Merck: Employee, Salary. S. Patel, Merck: Employee, Salary. D. Nahas, merck: Employee, Salary. M. Citron, merck: Employee, Salary. P. Cejas, merck: Employee, Salary. R. Swoyer, Merck: Employee, Salary. B. Luo, merck: Employee, Salary. M. Eddins, Merck: Employee, Salary. J. Reid, Merck: Employee, Salary. A. Fridman, Merck: Employee, Salary. J. Galli, Merck: Employee, Salary. S. Cosmi, Merck: Independent Contractor, Salary. G. Dhanasekeran, Merck: Employee, Salary. Z. Wen, Merck: Employee, Salary. X. He, Merck: Employee, Salary. D. Wang, Merck: Employee, Salary. G. Heidecker, Merck: Employee, Salary. J. Flynn, Merck: Employee, Salary. J. Cook, merck: Employee, Salary. S. Soisson, Merck: Employee, Salary. D. Casimiro, Merck: Employee, Salary. A. Bett, Merck: Employee, Salary. W. Blair, Merck: Employee, Salary. D. Distefano, Merck: Employee, Salary. C. Haines, Merck: Employee, Salary.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research on novel targets against pain.Although originally identified as an oncogene, TrkA is linked to pain and elevated levels of NGF, the ligand for TrkA, are associated with chronic pain.Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief.Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases.The x-ray structures reveal a novel binding site outside the kinase active site that utilizes residues from the kinase domain and the juxtamembrane region.Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes.The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes.The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition.This highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information due to substantial differences in the interacting residues.These structures illustrate the flexibility of binding to sequences outside of, but adjacent to, the kinase domain of TrkA.This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins and has implications on obtaining selectivity for kinase targets in drug discovery.

Crystallographic analysis of human O-GlcNAc hydrolase (hOGA) fragments containing the catalytic domain, including structures in complex with known inhibitors, suggests that OGA is functional as a dimer and defines opportunities for structure-based drug design.