Perspectives on Applications of F-NMR in Fragment-Based Drug Discovery.

Fragment-based drug discovery is a powerful approach in drug discovery, applicable to a wide range of targets. This method enables the discovery of potent compounds that can modulate target functions, starting from fragment compounds that bind weakly to the targets. While biochemical, biophysical, and cell-based assays are commonly used to identify fragments, F-NMR spectroscopy has emerged as a powerful tool for exploring interactions between biomolecules and ligands.

H, N and C backbone resonance assignment of the N-terminal region of Zika virus NS4B protein in detergent micelles.

Zika virus has raised global concerns due to its link to microcephaly and Guillain-Barré syndrome in adults. One of viral nonstructural proteins-NS4B, an integral membrane protein, plays crucial roles in viral replication by interacting with both viral and host proteins, rendering it an attractive drug target for antiviral development. We purified the N-terminal region of ZIKV NS4B (NS4B NTD) and reconstituted it into detergent micelles.

Protocol to perform fragment screening using NMR spectroscopy.

Fragment-based drug design plays an important role in drug discovery. Protein-observed NMR experiments with isotopically labeled samples are used to probe target-ligand interactions and map the ligand-binding sites. Here, we present a protocol to perform fragment screening using NMR spectroscopy.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV.

Identification of small-molecule binding sites of a ubiquitin-conjugating enzyme-UBE2T through fragment-based screening.

UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using F-nuclear magnetic resonance (NMR) and validated the hits with H- N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer.

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified.

Targeted Degradation of XIAP is Sufficient and Specific to Induce Apoptosis in MYCN-overexpressing High-risk Neuroblastoma.

Unlabelled: XIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFβ signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells.

Backbone H, N and C resonance assignments for an E2 ubiquitin conjugating enzyme-UBE2T.

Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications.

Backbone H, N and C resonance assignments for dengue NS2B without the NS3 protease cofactor region in detergent micelles.

Dengue virus is an important human pathogen affecting people especially in tropical and subtropical regions. Its genome encodes seven non-structural proteins that are important for viral assembly and replication. Dengue NS2B is a membrane protein containing four transmembrane helices and involved in protein-protein interactions.

H, N and C resonance assignments of the Q61H mutant of human KRAS bound to GDP.

KRAS proteins are small GTPases binding to the cell membrane and playing important roles in signal transduction. KRAS proteins form complexes with GTP and GDP to result in active and inactive conformations favouring interactions with different proteins. Mutations in KRAS have impact on the GTPase activity and some mutants are related to certain types of cancers.

Structure-activity relationship studies of allosteric inhibitors of EYA2 tyrosine phosphatase.

Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors.

Secondary structures, dynamics, and DNA binding of the homeodomain of human SIX1.

Human sine oculis homeobox homolog (SIX) 1 contains a homeodomain (HD), which is important for binding to DNA. In this study, we carried out structural studies on the HD of human SIX1 using nuclear magnetic resonance (NMR) spectroscopy. Its secondary structures and dynamics in solution were explored.

Discovery of Covalent Inhibitors Targeting the Transcriptional Enhanced Associate Domain Central Pocket.

Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new "thiol conjugation assay" for identification of novel small molecules that bind to the TEAD central pocket.

Progress in Developing Inhibitors of SARS-CoV-2 3C-Like Protease.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral outbreak started in late 2019 and rapidly became a serious health threat to the global population. COVID-19 was declared a pandemic by the World Health Organization in March 2020.

Mechanisms of Action for Small Molecules Revealed by Structural Biology in Drug Discovery.

Small-molecule drugs are organic compounds affecting molecular pathways by targeting important proteins. These compounds have a low molecular weight, making them penetrate cells easily. Small-molecule drugs can be developed from leads derived from rational drug design or isolated from natural resources.

A Practical Perspective on the Roles of Solution NMR Spectroscopy in Drug Discovery.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study structures and dynamics of biomolecules under physiological conditions. As there are numerous NMR-derived methods applicable to probe protein-ligand interactions, NMR has been widely utilized in drug discovery, especially in such steps as hit identification and lead optimization. NMR is frequently used to locate ligand-binding sites on a target protein and to determine ligand binding modes.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-7.

Breakthroughs in Medicinal Chemistry [...

Insights into Structures and Dynamics of Flavivirus Proteases from NMR Studies.

Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design.

Identification and structural characterization of small molecule fragments targeting Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) NS2B-NS3 protease is a validated antiviral target as it is essential for maturation of viral proteins. However, its negatively charged active site hinders the development of orthosteric small-molecule inhibitors. Fragment-based drug discovery (FBDD) is a powerful tool to generate novel chemical starting points against difficult drug targets.

F-NMR in Target-based Drug Discovery.

Solution NMR spectroscopy plays important roles in understanding protein structures, dynamics and protein-protein/ligand interactions. In a target-based drug discovery project, NMR can serve an important function in hit identification and lead optimization. Fluorine is a valuable probe for evaluating protein conformational changes and protein-ligand interactions.

Backbone resonance assignment for the full length tRNA-(NG37) methyltransferase of Pseudomonas aeruginosa.

Bacterial tRNA (guanine37-N)-methyltransferase (TrmD) plays important roles in translation, making it an important target for the development of new antibacterial compounds. TrmD comprises two domains with the N-terminal domain binding to the S-adenosyl-L-methionine (SAM) cofactor and the C-terminal domain critical for tRNA binding. Bacterial TrmD is functional as a dimer.

Secondary structure and topology of the transmembrane domain of Syndecan-2 in detergent micelles.

Syndecans are single-span membrane proteins playing important roles in cell-cell and cell-matrix interactions. The transmembrane domain of syndecans is critical for signal transduction across the cell membrane. Here, the structure of the transmembrane domain of syndecan-2 in detergent micelles was investigated using solution NMR spectroscopy.

Applications of In-Cell NMR in Structural Biology and Drug Discovery.

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein⁻protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells.

Backbone resonance assignment for the N-terminal region of bacterial tRNA-(NG37) methyltransferase.

Bacterial tRNA (guanine-N)-methyltransferase (TrmD) is an important antibacterial target due to its essential role in translation. TrmD has two domains connected with a flexible linker. The N-terminal domain (NTD) of TrmD contains the S-adenosyl-L-methionine (SAM) cofactor binding site and the C-terminal domain is critical for tRNA binding.