Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation.

Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å.

PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication.

The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef.

A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer.

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1, and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC).

PROTAC-mediated Degradation of HIV-1 Nef Efficiently Restores Cell-surface CD4 and MHC-I Expression and Blocks HIV-1 Replication.

The HIV-1 Nef accessory factor is critical to the viral life cycle where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers.

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions.

Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor.

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms.

ATP-site inhibitors induce unique conformations of the acute myeloid leukemia-associated Src-family kinase, Fgr.

The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively.

Inhibitors of HIV-1 Nef-Mediated Activation of the Myeloid Src-Family Kinase Hck Block HIV-1 Replication in Macrophages and Disrupt MHC-I Downregulation.

HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity , providing a robust assay for chemical library screening.

Virtual-Partition Digital PCR for High-Precision Chromosomal Counting Applications.

Digital PCR (dPCR) is the gold-standard analytical platform for rapid high-precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platform's ability to address some clinical applications that require the simultaneous monitoring of 20-50 analytes per sample. Here, we present virtual-partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow.

Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4-6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens.

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes.

Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus.

A single β-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding.

The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners.

Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding.

The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein-protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3-SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef.

Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen.

Background: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules.

Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency.

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck.

Background: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity.

Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470.

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi.