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BioAssay: AID 463187

384-well Z-Lyte format Hck-Nef inhibitor HTS run at the PMLSC

Assay Provider: Dr. Thomas Smithgall, University of Pittsburgh, Department of Molecular Genetics and Biochemistry. ..more
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AID: 463187
Data Source: University of Pittsburgh Molecular Library Screening Center (MH083223 Targeting HIV-1 Nef with Small Molecules)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-09-01

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BioActive Compounds: 1495
Assay Provider: Dr. Thomas Smithgall, University of Pittsburgh, Department of Molecular Genetics and Biochemistry.

Introduction, background, and significance extracted from Dr. Smithgall#s RO3 Application MH083223

Introduction. The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef has no known catalytic activity, and functions by interacting with multiple host cell signaling proteins, including members of the Src protein-tyrosine kinase family. Previous work from our laboratory has shown that Nef binds and activates Hck, a Src family kinase (SFK) selectively expressed in macrophages, which are an essential HIV target cell and viral reservoir. Recently, we found that Nef also activates c-Src and Lyn by a similar mechanism, suggesting that Nef-SFK complexes are present in most HIV-infected cell types. Other work has established that knock-down of Hck expression blocks M-tropic HIV replication in primary human macrophages, and that homozygous-null hck mice show longer latency for Nef-induced AIDS-like disease. Taken together, these studies strongly implicate Nef-SFK protein complexes in AIDS pathogenesis and as rational targets for anti-HIV drug discovery.

Significance. Due to the emergence of HIV strains resistant to conventional anti-retroviral agents, there is an urgent need to identify and validate new HIV targets for the development of novel therapeutics. HIV accessory proteins in general, and the Nef protein in particular, have been suggested as possible therapeutic targets but to date no useful compounds have been reported that work via these proteins. Our hypothesis, supported by preliminary data, is that HIV Nef in complex with a host cell kinase (Hck) essential for disease progression represents a rational target for novel anti-HIV drug discovery. As described in more detail below, interaction with Nef may induce a novel conformation of Hck that may be addressable with small molecules. In addition, our assay has the potential to identify novel structures that interact directly with Nef itself, compromising its ability to recruit and activate Hck in the assay. Finally,in addition to the potential for new therapeutics, identification of compounds that selectively modify HIV Nef function will be invaluable tools to explore the function of this enigmatic protein.

Overview of Src family kinase structure and regulation. The focus of this application is molecular targeting of the protein complexes formed between HIV Nef, an essential AIDS progression factor, and Hck, a Src-family member expressed in macrophages (an important target cell for HIV) and other myeloid leukocytes. All Src kinases exhibit N-terminal sequences for lipid attachment (myristoylation, and in some cases, palmitoylation), a unique domain, SH3, SH2 and kinase domains, followed by a C-terminal negative regulatory tail. Lipid modification of Src family kinases promotes membrane localization, which is often essential for biological activity. The SH3 domain contributes to substrate recruitment and is critical for the regulation of kinase activity. SH3 domains bind with high affinity and specificity to target sequences rich in proline and other hydrophobic amino acids. These sequences form a polyproline type II (PPII) helix which associates with the hydrophobic surface of the SH3 domain. In the case of Hck, the SH3 domain provides the binding site for HIV Nef (see below). SH2 domains function in protein-protein interaction by virtue of their affinity for phosphotyrosine-containing sequences. In the context of Src, the SH2 domain also contributes to the negative regulation of kinase activity. Phosphorylation of a highly conserved tyrosine residue in the tail region induces intramolecular interaction with the SH2 domain, pushing the kinase into an inactive conformation. This closed form of the kinase is stabilized by an additional intramolecular interaction of the SH3 domain with a PPII helix formed by the linker connecting the SH2 and kinase domains. The SH3:linker interaction was first discovered in the X-ray crystal structures of the inactive forms of c-Src and Hck.The tail Tyr residues of SFKs are phosphorylated by a distinct regulatory kinase known as Csk (for C-terminal Src kinase) and a related kinase known as Chk12. Gene-knockout experiments strongly suggest that Csk is the master regulator of all Src family members. Without Csk, embryonic lethality is observed with a concomitant elevation in overall SFK activity. In contrast, individual knockouts of Yes, Hck, Fgr, and Blk show modest or no detectable phenotypes, suggesting functional compensation by other family members during development and in the adult. This point is important because it suggests that isoform selective SFK inhibitors may have utility in the treatment of AIDS without producing side effects related to suppression of normal myeloid development or function. Src family kinase activation by displacement of intramolecular interactions involving the SH2 and SH3 domains. Src-related kinases have been implicated in signal transduction by both cytokines and growth factors, and in some cases are required for cell cycle progression in response to factor treatment. The mechanism of SFK activation by growth factor receptor tyrosine kinases may involve SH2-dependent recruitment to the activated, autophosphorylated form of the receptor. Binding of the SH2 domain to the receptor may induce Src activation by displacing the negative regulatory tail, leading to phosphorylation of the receptor in some cases9,44. The mechanism of SFK activation by cytokine receptors is less clear, but may also involve recruitment to the activated, oligomeric form of the receptor. Recruitment of two or more kinase molecules into close proximity may allow for trans-phosphorylation, which is important for kinase activation. We recently demonstrated that HIV Nef (which is also oligomeric) activates Hck by a similar mechanism. Binding of SFKs to substrates or other molecules via their SH3 domains is also sufficient to induce kinase activation in some instances. Physiological examples include the focal adhesion protein p130 Cas46, the progesterone receptor3, and the Stat3 transcription factor. These studies have led to the idea that SH3-dependent recruitment of substrates to SFKs may induce transient kinase activation, substrate phosphorylation and release, followed by a return of the kinase to its inactive state. Work from our laboratory has shown that SH3-dependent binding of HIV-1 Nef causes sustained activation of Hck in vivo, and this effect is sufficient to induce biological responses (oncogenic transformation of fibroblasts and survival signaling in myeloid cells). The mechanism involves displacement of the SH3 domain from its intramolecular interaction with the SH2:kinase linker. Indeed, we have observed that mutagenesis of linker proline residues is also sufficient to stimulate Hck kinase activity, presumably via a similar mechanism. These findings support a central role for SH3 domains in the regulation of Hck and other SFKs and identify Nef-SFK complexes as possible therapeutic targets in HIV-infected cells (see below).

HIV Nef: Essential AIDS progression factor and SH3-binding protein for Hck, Lyn and c-Src. Nef is a small (25-30 kDa) myristoylated protein that, like SFKs, is associated with the plasma membrane. Nef is required for the high-titer replication of both HIV and SIV and is essential for the development of AIDS-like disease in non-human primates. HIV strains with defective nef alleles have been isolated from patients with long-term, non-progressive HIV infection, implicating Nef as a critical virulence factor for AIDS. Furthermore, targeted expression of Nef to the T-cell and macrophage compartments of transgenic mice induces a severe AIDS-like syndrome, illustrating an essential role for this protein in disease progression. While Nef has no known catalytic activity, it affects the functions of multiple classes of host cell proteins, including viral (CD4, CXCR4, CCR5) and immune (MHC-I) receptors, trafficking proteins, guanine nucleotide exchange factors, and protein kinases. Interactions of Nef with Hck and other SFKs are among the best understood in molecular terms. Nef binds to the SH3 domain of Hck with high affinity for an SH3-mediated interaction (Kd = 250 nM). Mutagenesis has established that one determinant of HIV-1Nef-SH3 interaction is the consensus PxxP motif, PQVPxR50. X-ray crystallographic and NMR structural studies of Nef-SH3 complexes show that these residues form the critical polyproline type II helix that contacts the SH3 hydrophobic surface. The Nef PxxP motif responsible for SH3 bindingis highly conserved among primary HIV isolates, and is essential for the high-titer replication of HIV in un-stimulated peripheral blood mononuclear cells in vitro, and a murine model of AIDS. The Nef PxxP motif is also required for MHC and CCR5/CXCR4 down regulation, suggestive of a link between SFKs and this important Nef function. Indeed, recent work from Thomas and co-workers shows that Nef uses its PxxP motif to assemble SFK-Syk-PI3K complexes essential for MHC-I downregulation. Work from our group has established that the PxxP motif is required for constitutive activation of Hck, Lyn and c-Src in vitro and in cell-based assays. Although the Nef PxxP motif is essential for Hck SH3 binding and kinase activation, structural analysis suggests that it is not the sole determinant of high-affinity binding. Also essential are residues from the Nef #A and #B helices, which form a hydrophobic pocket that accommodates an Ile residue found in the RT loop of the SH3 domain. This RT loop Ile residue is unique to the Hck and Lyn SH3 domains, providing a structural basis for an important relationship between Nef and these SFKs in HIV pathogenesis. Recently, we tested the importance of the conserved hydrophobic pocket residue Tyr 120 in Nef-Hck complex formation and signaling. Mutagenesis of this single residue almost completely abolished Nef-Hck interaction and Hck activation by Nef in vivo. These data suggest that small molecules targeted to this pocket may affect Nef:SFK interaction and downstream signaling.

Nef, Src family kinases, and HIV/AIDS. Structural and biochemical data summarized above argue strongly that the SH3-binding function of Nef in general and its interaction with Hck and other SFKs in particular are critical to its functions in HIV replication and AIDS progression. Recent data with the Nef transgenic mouse model also support this view. In a follow-up to their original work, Hanna et al, showed that transgenic mice expressing Nef in which the prolines in the PxxP SH3-binding motif are mutated to alanines never develop AIDS-like disease. They also crossed the wild-type Nef mice with mice in which both Hck alleles were deleted by gene targeting. The resulting Nef+/Hck-/- animals showed a dramatic delay in the induction of this Nef-induced pathology. In addition, while none of the control Nef transgenics survived past 9 months, nearly 25% of the Nef+/Hck-/- animals were alive after 12 months. These data provide strong genetic evidence that both the Nef PxxP motif and Nef-Hck interaction are essential for AIDS progression. Moreover, they identify the Nef-Hck complex and Nef itself as rational targets for drug discovery. An essential role for Hck in HIV replication in macrophages is suggested by recent work from Komuro, et al. In this study, human macrophages cultured in the presence of M-CSF were found to express high levels of Hck and were permissive for M-tropic HIV replication. Suppression of Hck expression with antisense oligonucleotides dramatically inhibited viral replication, directly supporting a role for Hck in viral replication. Because macrophages are a major site of Hck expression in vivo, constitutive activation of Hck by Nef in macrophages seems likely to account for some aspect of the known effects of Nef in AIDS progression. Most primary HIV isolates replicate efficiently in primary monocytes and macrophages. During initial exposure to HIV, M-tropic strains promote viral spread by inducing chemokine release from macrophages and attracting uninfected T cells; this effect is dependent in part upon Nef. In the brain, HIV-infected microglial cells, the resident macrophages of the CNS, release a variety of neurotoxic substances which may contribute to development of AIDS dementia48. Macrophages are also important sites for viral replication in end-stage disease, possibly explaining the high viral loads observed even after T-cell depletion. Activation of Hck by Nef may also contribute to AIDS progression by promoting survival of HIV-infected macrophages and their progenitors. Indeed, we have observed that Nef protects macrophage progenitor cells from apoptosis through a distinct pathway involving induction of Bcl-XL.

Nef-Hck HTS Assay
The assay selected for the primary HTS utilized the Z#-Lyte method (Invitrogen), which employs a fluorescence-based, coupled-enzyme format. The assay takes advantage of the differential sensitivity of phosphorylated and non-phosphorylated peptide substrates to proteolytic cleavage. In the primary reaction, the kinase phosphorylates a single tyrosine residue in the synthetic FRET-peptide (Tyr2 peptide in our case). The kinase reactions are then developed with a site-specific protease which selectively cleaves the non-phosphorylated FRET-peptide. Cleavage prevents FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the N- and C-terminii of the FRET-peptide, whereas the uncleaved phosphopeptides maintain a FRET signal. Activity is expressed as an #Emission Ratio# of donor to acceptor emission after excitation of the donor fluorophore at 400 nm: donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm. Both cleaved and uncleaved FRET-peptides contribute to the fluorescence signals and therefore to the Emission Ratio. The Emission Ratio will remain low if the FRET-peptide is phosphorylated (i.e., no kinase inhibition) and will be high if the FRET-peptide is non-phosphorylated (i.e., kinase inhibition). Using the Z#-Lyte assay, we first established assay conditions under which Hck-YEEI activation was dependent upon the presence of Nef. Peptide substrate phosphorylation increased as a function of the amount of Hck-YEEI added to the assay. In the presence of a 10-fold molar excess of HIV-1 Nef ateach Hck-YEEI concentration, the presence of Nef markedly shifted the Hck-YEEI activation curve to the left, indicative of its ability to bind to Hck and relieve the inhibitory effect of the SH3 domain on kinase activity as reported previously. The assay is quite sensitive: Hck activity can be detected (with Nef present) using as little as 5-10 ng of Hck/well. The primary HTS was run under conditions where Hck activation was dependent upon Nef.
HIV Hck:Nef HTS Protocol
1. Recombinant Hck-YEEI protein, was purified to homogeneity from Sf-9 insect cells
2. Recombinant HIV-1 Nef (SF2 strain), was purified to homogeneity from E. coli
3. Z-Lyte assay reagents were purchased from (Invitrogen).

Several Hck and Nef protein preparations provided by the assay provider were utilized in the HTS campaign, and several Hck:Nef protein ratios were utilized 10 ng + 50 ng, 12.5 ng + 75 ng, 15 ng + 75 ng, and 20 ng + 100 ng in the 384-well HTS assay.

HTS Protocol:
1. Thaw compound plates, 2uL of 1 mM in 100% DMSO.
2. Spin compound plates down, 5 min 50 x g.
3. Dilute 2 uL 1mM compounds with 23ul of water on Flex Drop (80 uM, 8% DMSO).
4. Mix and Transfer 2.5ul of compound to assay plate on EP3.
5. Transfer 5ul of Max (2.5ul DMSO and 2.5ul Hck:Nef) and Min (2.5ul DMSO and 2.5ul Hck/1x kinase) controls (from pre-made control plates) to assay plate on EP3.
6. Add 2.5ul of Hck:Nef to assay plate(340 wells), spin down and incubate for 30 min at ambient temperature (40 uM, 4% DMSO).
7. Add 5 uL of ATP/Tyr complex to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 50 min (20 uM, 2% DMSO).
8. Add Development buffer to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 60 min.
9. Add Stop reagent to whole plate on MicroFlow, spin down.
10. Read Donor to Acceptor FRET Emission Ratio on M5 plate reader within 2 hrs. Excitation of the donor fluorophore at 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm.
Hck-Nef Assay HTS Activity scoring rules:
The 384-well format Hck-Nef inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% inhibition to identify active compounds.
Max control: 2.5ul DMSO and 2.5ul Hck:Nef
Min control: 2.5ul DMSO and 2.5ul Hck/1x kinase (No Nef)
Hck-Nef Inhibitor scoring rules:
1 - Substance is considered inactive when the % inhibition is < 50 %
2 - Substance is considered active when % inhibition is >/= 50 %
3 - Substance activity outcome is inconclusive
0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 50 %, the score is 40.
b) if the % inhibition is < 50 %, the score is 0.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1HTS raw dataDonor to acceptor#FRET emission Ratio#. Excitation of the donor fluorophore at 400 nm, donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm.Floatratio
2HTS % Inhibition (20μM**)% inhibition of the Hck-Nef assay at 20 uM calculated from the mean max and min plate controlsFloat%
3Mean max signalMean Donor to acceptor FRET emission Ratio. Excitation 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm of maximum assay signal window plate controls n=32Floatratio
4Mean min signalMean Donor to acceptor FRET emission Ratio. Excitation 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm of minimum assay signal window plate controls n=24Floatratio
5Assay plate Z-factorZ'-factor calculated from the maximum and mimimum assay signal window plate controlsFloat
6Assay plate S:BSignal to background ratio calculated from the means of the maximum and mimimum assay signal window plate controls Float
7HTS Assay DateDate the assay was perfomed by the PMLSCString

** Test Concentration.
Additional Information
Grant Number: MH083223

Data Table (Concise)
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