ABSTRACT
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , Ligands , Polyubiquitin/metabolism , Rats , ThermodynamicsABSTRACT
Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.
Subject(s)
Ceramides/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Multiple Sclerosis/drug therapy , Serine C-Palmitoyltransferase/metabolism , Sphingosine/toxicity , Animals , Disease Models, Animal , Fingolimod Hydrochloride/pharmacology , Humans , Myelin Sheath/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Rats , Spinal Cord/metabolismABSTRACT
Cellular junctions are essential to the normal functioning of the endothelium and control angiogenesis, tissue leak, and inflammation. From a screen of micro RNAs (miRNAs) altered in in vitro angiogenesis, we selected a subset predicted to target junctional molecules. MiR-27a was rapidly downregulated upon stimulation of in vitro angiogenesis, and its level of expression is reduced in neovessels in vivo. The downregulation of miR-27a was essential for angiogenesis because ectopic expression of miR-27a blocked capillary tube formation and angiogenesis. MiR-27a targets the junctional, endothelial-specific cadherin, VE-cadherin. Consistent with this, vascular permeability to vascular endothelial growth factor in mice is reduced by administration of a general miR-27 inhibitor. To determine that VE-cadherin was the dominant target of miR-27a function, we used a novel technology with "Blockmirs," inhibitors that bind to the miR-27 binding site in VE-cadherin. The Blockmir CD5-2 demonstrated specificity for VE-cadherin and inhibited vascular leak in vitro and in vivo. Furthermore, CD5-2 reduced edema, increased capillary density, and potently enhanced recovery from ischemic limb injury in mice. The Blockmir technology offers a refinement in the use of miRNAs, especially for therapy. Further, targeting of endothelial junctional molecules by miRNAs has clinical potential, especially in diseases associated with vascular leak.
Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Gene Expression Regulation , MicroRNAs/metabolism , Animals , Binding Sites , Capillary Permeability , Edema/pathology , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Ischemia/pathology , Liver Cirrhosis/pathology , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , Neovascularization, PathologicABSTRACT
We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.
ABSTRACT
Observations of male alternative reproductive tactics (ARTs) in a variety of species have stimulated the development of mathematical models that can account for the evolution and stable coexistence of multiple male phenotypes. However, little attention has been given to the population dynamic consequences of ARTs. We present a population model that takes account of the existence of two male ARTs (guarders and sneakers), assuming that tactic frequencies are environmentally determined and tactic reproductive success depends on the densities of both types. The presence of sneakers typically increases overall population density. However, if sneakers comprise a sufficiently large proportion of the population-or, equivalently, if guarders are sufficiently rare-then it is possible for the total population to crash to extinction (in this extreme regime, there is also an Allee effect, i.e. a threshold density below which the population will go extinct). We apply the model to the example of the invasive round goby (Neogobius melanostomus). We argue that ARTs can dramatically influence population dynamics and suggest that considering such phenotypic plasticity in population models is potentially important, especially for species of conservation or commercial importance.
Subject(s)
Perciformes , Reproduction , Animals , Male , Population Density , Population Dynamics , Sexual Behavior, AnimalABSTRACT
The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKε and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.
Subject(s)
Fas-Associated Death Domain Protein/metabolism , Interferon-alpha/biosynthesis , Respirovirus Infections/metabolism , Ribonucleoproteins/metabolism , Sendai virus/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Dogs , Fas-Associated Death Domain Protein/genetics , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza, Human/genetics , Influenza, Human/metabolism , Interferon Regulatory Factor-7/genetics , Interferon Regulatory Factor-7/metabolism , Interferon-alpha/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Respirovirus Infections/genetics , Ribonucleoproteins/genetics , Sendai virus/genetics , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination/geneticsABSTRACT
Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.
Subject(s)
Drug Resistance, Multiple, Bacterial/drug effects , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Monobactams/pharmacology , Animals , Binding Sites/drug effects , Blood Proteins/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Gram-Negative Bacterial Infections/microbiology , Humans , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Monobactams/chemical synthesis , Monobactams/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Synthetic approaches to mixed ligand chelators on readily available t-butylphenol-formaldehyde oligomer, PFO, scaffolds were examined. In a promising approach, tris and tetraphenol oligomers were selectively mono or di protected using t-butyldiphenyl silyl chloride. The utility of these protected intermediates to prepare representative mixed PFO chelators, carrying ligands such as hydroxamic acid, 3,2-hydroxypyridinones and others was then demonstrated. The introduction of the ligand tethers onto the phenolic scaffold can be done sequentially under relatively mild conditions that tolerate the presence of other sensitive ligand groups. The differential reactivity of the disilyl derivative 20b, allowed stepwise introduction of two different ligands on the internal phenolic positions. This enabled the introduction of three different ligand groups of choice onto the tetra phenol platform.
ABSTRACT
The polyethylene glycol (PEG) moiety has become increasingly important in medicinal chemistry. Herein, we describe the PEG functionalization of amines via hydrogen borrowing reductive amination. This was accomplished using the [Ru(p-cymene)Cl2]2 catalyst and phosphorus-containing ligand dppf or DPE to yield a variety of PEGylated primary and secondary amine products. Furthermore, we illustrate the utility of this method with the synthesis of quetiapine (Seroquel) in 62% isolated yield.
ABSTRACT
T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR zeta subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-kappaB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogenes were equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations.
Subject(s)
NF-kappa B/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 4/physiology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/physiology , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Humans , Jurkat Cells , Kidney/cytology , Mice , NF-kappa B/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 4/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 4/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 4/metabolism , Receptors, Antigen, T-Cell/immunology , TransfectionABSTRACT
Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.
Subject(s)
Interleukin-17/antagonists & inhibitors , Interleukin-17/chemistry , Peptides, Cyclic/pharmacology , Small Molecule Libraries/pharmacology , Algorithms , Binding Sites , Cells, Cultured , Drug Design , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Molecular Dynamics Simulation , Peptides, Cyclic/chemistry , Protein Binding , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
During meiosis DNA double-strand breaks initiate recombination in the distantly related budding and fission yeasts and perhaps in most eukaryotes. Repair of broken meiotic DNA is essential for formation of viable gametes. We report here distinct but overlapping sets of proteins in these yeasts required for formation and repair of double-strand breaks. Meiotic DNA breakage in Schizosaccharomyces pombe did not require Rad50 or Rad32, although the homologs Rad50 and Mre11 are required in Saccharomyces cerevisiae; these proteins are required for meiotic DNA break repair in both yeasts. DNA breakage required the S. pombe midmeiosis transcription factor Mei4, but the structurally unrelated midmeiosis transcription factor Ndt80 is not required for breakage in S. cerevisiae. Rhp51, Swi5, and Rad22 + Rti1 were required for full levels of DNA repair in S. pombe, as are the related S. cerevisiae proteins Rad51, Sae3, and Rad52. Dmc1 was not required for repair in S. pombe, but its homolog Dmc1 is required in the well-studied strain SK1 of S. cerevisiae. Additional proteins required in one yeast have no obvious homologs in the other yeast. The occurrence of conserved and nonconserved proteins indicates potential diversity in the mechanism of meiotic recombination and divergence of the machinery during the evolution of eukaryotes.
Subject(s)
Conserved Sequence , DNA Repair/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Yeasts/genetics , DNA Damage , Meiosis/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/cytology , Schizosaccharomyces/geneticsABSTRACT
Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.
Subject(s)
Fas-Associated Death Domain Protein/physiology , Granulocytes/pathology , Inflammation/pathology , Macrophages/pathology , Myeloid Differentiation Factor 88/physiology , Receptor-Interacting Protein Serine-Threonine Kinases/physiology , Animals , Apoptosis , Blotting, Western , Dendritic Cells/metabolism , Dendritic Cells/pathology , Enzyme-Linked Immunosorbent Assay , Female , Granulocytes/metabolism , Inflammation/etiology , Inflammation/metabolism , Integrases/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Necrosis , Signal Transduction , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
Reported here are procedures for a one-pot oxidation and rearrangement of propargylamines to synthesize enaminones, with supporting mechanistic studies. Also reported are the extended one-pot syntheses of pyrazoles, including celecoxib and various heterocyclic compounds.
Subject(s)
Pargyline/analogs & derivatives , Propylamines/chemistry , Pyrazoles/chemical synthesis , Sulfonamides/chemical synthesis , Catalysis , Celecoxib , Combinatorial Chemistry Techniques , Molecular Structure , Oxidation-Reduction , Pargyline/chemistry , Pyrazoles/chemistry , Sulfonamides/chemistryABSTRACT
Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.
Subject(s)
Gram-Negative Bacteria/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Siderophores/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Microbial Sensitivity Tests , Peptides/drug effects , Peptides, Cyclic , beta-Lactamases/metabolismABSTRACT
The relationship between dendritic cells (DCs) and commensal microflora in shaping systemic immune responses is not well understood. Here, we report that mice deficient for the Fas-associated death domain in DCs developed systemic inflammation associated with elevated proinflammatory cytokines and increased myeloid and B cells. These mice exhibited reduced DCs in gut-associated lymphoid tissues due to RIP3-dependent necroptosis, whereas DC functions remained intact. Induction of systemic inflammation required DC necroptosis and commensal microbiota signals that activated MyD88-dependent pathways in other cell types. Systemic inflammation was abrogated with the administration of broad-spectrum antibiotics or complete, but not DC-specific, deletion of MyD88. Thus, we have identified a previously unappreciated role for commensal microbiota in priming immune cells for inflammatory responses against necrotic cells. These studies demonstrate the impact intestinal microflora have on the immune system and their role in eliciting proper immune responses to harmful stimuli.
Subject(s)
Dendritic Cells/immunology , Microbiota/physiology , Necrosis/metabolism , Animals , Dendritic Cells/metabolism , Inflammation/immunology , Inflammation/metabolism , Mice , Signal TransductionABSTRACT
Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.
Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Monobactams/pharmacology , Pyridones/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Escherichia coli/drug effects , Inhibitory Concentration 50 , Klebsiella pneumoniae/drug effects , Male , Microbial Sensitivity Tests , Molecular Structure , Monobactams/chemistry , Monobactams/pharmacokinetics , Pseudomonas aeruginosa/drug effects , Pyridones/chemistry , Pyridones/pharmacokinetics , Rats , Rats, WistarABSTRACT
The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.
Subject(s)
CD3 Complex/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Signal Transduction/physiology , beta-Adrenergic Receptor Kinases/metabolism , Animals , CD3 Complex/genetics , G-Protein-Coupled Receptor Kinase 2 , Humans , Jurkat Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Receptors, G-Protein-Coupled/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolism , beta-Adrenergic Receptor Kinases/geneticsABSTRACT
Providing care to neonates with bowel obstruction requires a basic understanding of gastrointestinal (GI) anatomy and functional landmarks as well as knowledge of the pathophysiology associated with intestinal blockage. Early recognition and prompt diagnosis necessitate astute assessment of common presenting symptoms and accurate interpretation of diagnostic investigations. Initial medical management is focused primarily on gastric decompression and maintenance of fluid and electrolyte balance. This article describes features of the neonatal GI tract and discusses common causes of neonatal bowel obstruction.
Subject(s)
Digestive System Abnormalities , Intestinal Obstruction , Digestive System Abnormalities/diagnosis , Digestive System Abnormalities/physiopathology , Digestive System Abnormalities/therapy , Humans , Infant, Newborn , Intestinal Obstruction/diagnosis , Intestinal Obstruction/physiopathology , Intestinal Obstruction/therapyABSTRACT
Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.