An Allosteric Mechanism for Inhibiting HIV-1 Integrase with a Small Molecule

HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN

HIV-1 Integrase-Targeted Short Peptides Derived from a Viral Protein R Sequence

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69–75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site

Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.National Institutes of Health (U.S.) (Grants ES015339 and GM104047

HIV Integrase Inhibitors Block Replication of Alpha-, Beta-, and Gammaherpesviruses

ABSTRACT The catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activity in vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activity in vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi’s sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses

Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 Gag PTAP Interaction

SummaryBudding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.4–1.6 Å structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions

Synchronization of Isolated Downstates (K-Complexes) May Be Caused by Cortically-Induced Disruption of Thalamic Spindling

Sleep spindles and K-complexes (KCs) define stage 2 NREM sleep (N2) in humans. We recently showed that KCs are isolated downstates characterized by widespread cortical silence. We demonstrate here that KCs can be quasi-synchronous across scalp EEG and across much of the cortex using electrocorticography (ECOG) and localized transcortical recordings (bipolar SEEG). We examine the mechanism of synchronous KC production by creating the first conductance based thalamocortical network model of N2 sleep to generate both spontaneous spindles and KCs. Spontaneous KCs are only observed when the model includes diffuse projections from restricted prefrontal areas to the thalamic reticular nucleus (RE), consistent with recent anatomical findings in rhesus monkeys. Modeled KCs begin with a spontaneous focal depolarization of the prefrontal neurons, followed by depolarization of the RE. Surprisingly, the RE depolarization leads to decreased firing due to disrupted spindling, which in turn is due to depolarization-induced inactivation of the low-threshold Ca2+ current (IT). Further, although the RE inhibits thalamocortical (TC) neurons, decreased RE firing causes decreased TC cell firing, again because of disrupted spindling. The resulting abrupt removal of excitatory input to cortical pyramidal neurons then leads to the downstate. Empirically, KCs may also be evoked by sensory stimuli while maintaining sleep. We reproduce this phenomenon in the model by depolarization of either the RE or the widely-projecting prefrontal neurons. Again, disruption of thalamic spindling plays a key role. Higher levels of RE stimulation also cause downstates, but by directly inhibiting the TC neurons. SEEG recordings from the thalamus and cortex in a single patient demonstrated the model prediction that thalamic spindling significantly decreases before KC onset. In conclusion, we show empirically that KCs can be widespread quasi-synchronous cortical downstates, and demonstrate with the first model of stage 2 NREM sleep a possible mechanism whereby this widespread synchrony may arise

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in hela cells

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.National Institutes of Health (U.S.) (Grants ES015339 and GM104047

Phencyclidine-induced catalepsy in pigeons: Specificity and stereoselectivity

A procedure is described for the rapid assessment of cataleptic activity (loss of righting, without head-drop and without eye closure) of phencyclidine-type drugs. Single- and cumulative-dosing procedures with phencyclidine and ketamine produced similar results. Pentobarbital produced loss of righting at doses which also induced head-drop and eye closure. Catalepsy was induced exclusively by the d-isomers of ketamine, 1-(1-phenylcyclohexyl)-3-methylpiperidine and [alpha]-dioxadrol. The procedure is suitable for studying compounds which may interact with phencyclidine receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24637/1/0000048.pd

Nucleic Acids Res

Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed

Development of Grb2 SH2 Domain Signaling Antagonists: A Potential New Class of Antiproliferative Agents

Aberrant signaling through protein-tyrosine kinase (PTK)-dependent pathways is associated with several proliferative diseases. Accordingly, PTK inhibitors are being developed as new approaches for the treatment of certain cancers. Growth factor receptor bound protein 2 (Grb2) is an important downstream mediator of PTK signaling that serves obligatory roles in many pathogenic processes. One of the primary functions of Grb2 is to bind to specific phosphotyrosyl (pTyr)-containing sequences through its Src homology 2 (SH2) domain. Agents that bind to the Grb2 SH2 domain and prevent its normal function could disrupt associated PTK signaling and serve as alternatives to kinase-directed inhibitors. Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these efforts. The presentation will be thematically arranged according to the region of peptide modified, proceeding from the N-terminus to the C-terminus, with a special section devoted to aspects of conformational constraint