Karyn McFadden

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120.

In this work, we identified a high affinity and potency metallocene‐containing triazole peptide conjugate that suppresses the interactions of HIV‐1 envelope gp120 at both its CD4 and co‐receptor binding sites. The ferrocene‐peptide conjugate, HNG‐156, was formed by an on‐resin copper‐catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl‐containing triazole conjugate HNG‐105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV‐1 gp120 due mainly to the decreased dissociation rate of the conjugate‐gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037‐08) and clade B (YU‐2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV‐1YU‐2 gp120 at both the CD4 binding site and CD4‐induced binding sites. HNG‐156 showed a close‐to nanomolar IC50 for inhibiting cell infection by HIV‐1BaL whole virus. The dual receptor site antagonist activity and potency of HNG‐156 make it a promising viral envelope inhibitor lead for developing anti‐HIV‐1 treatments. Copyright

Interplay between DNA tumor viruses and the host DNA damage response.

Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposis sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.

The Active Core in a Triazole Peptide Dual‐Site Antagonist of HIV‐1 gp120

In an effort to identify broadly active inhibitors of HIV‐1 entry into host cells, we previously reported a family of dodecamer triazole–peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co‐receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG‐156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy‐ and amino‐terminal residues from the parent 12‐residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)‐Trp cluster at residues 6 and 7 with ferrocenyl‐triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino‐terminal residues distal to the central triazole(Pro)‐Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3–7 (Asn‐Asn‐Ile‐Ftp‐Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)‐Trp cluster, with a short N‐terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV‐1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV‐1 envelope that could be targeted for small‐molecule inhibitor discovery.

Metabolic stress is a barrier to Epstein–Barr virus-mediated B-cell immortalization

Significance Epstein–Barr virus (EBV) was the first human tumor virus discovered. Although nearly all adults are infected with EBV, very few go on to develop disease, for reasons that we are only beginning to understand. Infection with EBV induces a period of very rapid cell division, which requires an increased supply of metabolites, such as nucleotides, amino acids, and lipids. We found that EBV-infected cells that are unable to meet this increased metabolic demand are forced to stop proliferating and undergo a permanent growth arrest called senescence. Epstein–Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.

Antiviral Breadth and Combination Potential of Peptide Triazole HIV-1 Entry Inhibitors

ABSTRACT The first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection.

Cell-free HIV-1 virucidal action by modified peptide triazole inhibitors of Env gp120.

Initial entry of HIV-1 into host cells remains a compelling and yet elusive target for developing agents to prevent infection. This step is mediated by a sequence of interactions of a trimeric gp120/gp41 envelope (Env) protein complex with host cells, including initial gp120 encounter with the cellular receptor CD4 and a chemokine co-receptor usually either CCR5 or CXCR4 [1]. A peptide triazole class of entry inhibitor leads has been shown to bind to gp120 with close to nanomolar affinity, to suppress protein ligand interactions of the Env protein at both its CD4 and co-receptor binding sites and to inhibit cell infection by a broad range of virus subtypes [2]. These inhibitors appear to function mechanistically by conformationally entrapping gp120 in an inactivated state, different from either the flexible ground state of gp120 or the highly structured CD4-activated state. This entrapment effectively halts the entry process at the initial binding stages.

Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

BackgroundWe examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity.ResultsKR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions.ConclusionsThe antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion.

A recombinant allosteric lectin antagonist of HIV-1 envelope gp120 interactions.

The first, critical stage of HIV‐1 infection is fusion of viral and host cellular membranes initiated by a viral envelope glycoprotein gp120. We evaluated the potential to form a chimeric protein entry inhibitor that combines the action of two gp120‐targeting molecules, an allosteric peptide inhibitor 12p1 and a higher affinity carbohydrate‐binding protein cyanovirin (CVN). In initial mixing experiments, we demonstrated that the inhibitors do not interfere with each other and instead show functional synergy in inhibiting viral cell infection. Based on this, we created a chimera, termed L5, with 12p1 fused to the C‐terminal domain of CVN through a linker of five penta‐peptide repeats. L5 revealed the same broad specificity as CVN for gp120 from a variety of clades and tropisms. By comparison to CVN, the L5 chimera exhibited substantially increased inhibition of gp120 binding to receptor CD4, coreceptor surrogate mAb 17b and gp120 antibody F105. These binding inhibition effects by the chimera reflected both the high affinity of the CVN domain and the allosteric action of the 12p1 domain. The results open up the possibility to form high potency chimeras, as well as noncovalent mixtures, as leads for HIV‐1 envelope antagonism that can overcome potency limits and potential virus mutational resistance for either 12p1 or CVN alone. Proteins 2007.

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest.

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

HIV-1 Env gp120 Structural Determinants for Peptide Triazole Dual Receptor Site Antagonism

Despite advances in HIV therapy, viral resistance and side‐effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV‐1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co‐receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptides affinity and its ability to inhibit binding of CD4 and the co‐receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single‐round cell infection assays. Two mutations, D474A and T257A, caused large‐scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co‐receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development. Proteins 2013.