Pharmacokinetics of two common antiretroviral regimens in older HIV-infected patients: a pilot study.

OBJECTIVES: The pharmacokinetics (PK) of antiretrovirals (ARVs) in older HIV-infected patients are poorly described. Here, the steady-state PK of two common ARV regimens [tenofovir (TFV)/emtricitabine (FTC)/efavirenz (EFV) and TFV/FTC/atazanavir (ATV)/ritonavir (RTV)] in older nonfrail HIV-infected patients are presented. METHODS: HIV-infected subjects ≥ 55 years old not demonstrating the frailty phenotype were enrolled in an unblinded, intensive-sampling PK study. Blood plasma (for TFV, FTC, EFV, ATV and RTV concentrations) and peripheral blood mononuclear cells [PBMCs; for tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) concentrations] were collected at 11 time-points over a 24-hour dosing interval. Drug concentrations were analysed using validated liquid chromatography-ultraviolet detection (LC-UV) or liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. Noncompartmental pharmacokinetic analysis was used to estimate PK parameters [area under the concentration-time curve over 24 h (AUC0-24h ) and maximal concentration (Cmax )]. These parameters were compared with historical values from the general HIV-infected population. RESULTS: Six subjects on each regimen completed the study. Compared with the general population, these elderly subjects had 8-13% decreased TFV AUC0-24h and Cmax , and 19-78% increased FTC and RTV AUC0-24h and Cmax . Decreased ATV AUC0-24h (12%) and increased Cmax (9%) were noted, while EFV exposure was unchanged (5%) with a 16% decrease in Cmax . Intracellular nucleoside/tide metabolite concentrations and AUC are also reported for these subjects. CONCLUSIONS: This study demonstrates that the PK of these ARVs are altered by 5-78% in an older HIV-infected population. Implications of PK differences for clinical outcomes, particularly with the active nucleoside metabolites, remain to be explored. This study forms the basis for further study of ARV PK, efficacy, and toxicity in older HIV-infected patients.

Histone H3 E50K mutation confers oncogenic activity and supports an EMT phenotype.

Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype in human cells. Expression of H3E50K is sufficient to transform human cells as evidenced by a dramatic increase in cell migration and invasion, and a statistically significant increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K perturbs proximal H3 post-translational modifications globally and dysregulates gene expression, activating the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional histone H3 mutations have the potential to be oncogenic drivers and function through distinct mechanisms that dysregulate gene expression.

Dynamic localization of the nuclear import receptor and its interactions with transport factors.

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi-intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.

Nucleocytoplasmic transport of macromolecules.

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.

Interaction between the small GTPase Ran/Gsp1p and Ntf2p is required for nuclear transport.

Bidirectional movement of proteins and RNAs across the nuclear envelope requires Ran, a Ras-like GTPase. A genetic screen of the yeast Saccharomyces cerevisiae was performed to isolate conditional alleles of GSP1, a gene that encodes a homolog of Ran. Two temperature-sensitive alleles, gsp1-1 and gsp1-2, were isolated. The mutations in these two alleles map to regions that are structurally conserved between different members of the Ras family. Each mutant strain exhibits various nuclear transport defects. Both biochemical and genetic experiments indicate a decreased interaction between Ntf2p, a factor which is required for protein import, and the mutant GSP1 gene products. Overexpression of NTF2 can suppress the temperature sensitive phenotype of gsp1-1 and gsp1-2 and partially rescue nuclear transport defects. However, overexpression of a mutant allele of NTF2 with decreased binding to Gsp1p cannot rescue the temperature sensitivity of gsp1-1 and gsp1-2. Taken together, these data demonstrate that the interaction between Gsp1p and Ntf2p is critical for nuclear transport.

Mms22p protects Saccharomyces cerevisiae from DNA damage induced by topoisomerase II.

The cleavage reaction of topoisomerase II, which creates double-stranded DNA breaks, plays a central role in both the cure and initiation of cancer. Therefore, it is important to understand the cellular processes that repair topoisomerase II-generated DNA damage. Using a genome-wide approach with Saccharomyces cerevisiae, we found that Deltamre11, Deltaxrs2, Deltarad50, Deltarad51, Deltarad52, Deltarad54, Deltarad55, Deltarad57 and Deltamms22 strains were hypersensitive to etoposide, a drug that specifically increases levels of topoisomerase II-mediated DNA breaks. These results confirm that the single-strand invasion pathway of homologous recombination is the major pathway that repairs topoisomerase II-induced DNA damage in yeast and also indicate an important role for Mms22p. Although Deltamms22 strains are sensitive to several DNA-damaging agents, little is known about the function of Mms22p. Deltamms22 cultures accumulate in G2/M, and display an abnormal cell cycle response to topoisomerase II-mediated DNA damage. MMS22 appears to function outside of the single-strand invasion pathway, but levels of etoposide-induced homologous recombination in Deltamms22 cells are lower than wild-type. MMS22 is epistatic with RTT101 and RTT107, genes that encode its protein binding partners. Finally, consistent with a role in DNA processes, Mms22p localizes to discrete nuclear foci, even in the absence of etoposide or its binding partners.

The interaction between Ran and NTF2 is required for cell cycle progression.

The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus. Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly. In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state. Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus. Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle. A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature. Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G(2) in a MAD2 checkpoint-dependent manner. These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo. Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest.

Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis.

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.

Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4'-(9-acridinylamino)methane-sulfon-m-anisidide.

The effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.

Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran.

Nuclear transport factor 2 (NTF2) is associated with the translocation stage of nuclear protein import and binds both to nuclear pore proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in nuclear protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule's hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell nuclear protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient nuclear protein import.

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.

Identification and characterization of the human MOG1 gene.

Many of the proteins that mediate transport into and out of the nucleus have been structurally and functionally conserved throughout evolution. Here we describe the sequence and characterization of the human MOG1 gene. The MOG1 gene was originally identified in Saccharomyces cerevisiae as a multi-copy suppressor of conditional alleles of the yeast nuclear transport factor, GSP1 (scRan) (Oki and Nishimoto (1998) Proc. Natl. Acad. Sci. USA 95, 15388-15393). A search of the expressed sequence tag database identified a putative human protein that is 29% identical and 47% similar to the yeast protein. Our experiments demonstrate that the human MOG1 message is expressed in a variety of tissue samples. Several experiments indicate that the human MOG1 protein binds to both yeast and human Ran suggesting functional conservation between the yeast and human MOG1 proteins. Furthermore, hMOG1a, like scMOG1, is localized throughout the cell but is concentrated within the nucleus. Consistent with these findings, hMOG1a can partially complement the growth defect present in yeast MOG1 deletion cells. Taken together, our findings suggest that MOG1 is an evolutionarily conserved Ran binding protein that could play a role in regulating nuclear protein trafficking.

SGD1 encodes an essential nuclear protein of Saccharomyces cerevisiae that affects expression of the GPD1 gene for glycerol 3-phosphate dehydrogenase.

We here report the identification of the previously uncharacterized SGD1 gene, encoding a 102.8-kDa protein containing a leucine zipper region and a bipartite nuclear localization signal. Deletion of SGD1 results in loss of cell viability, while an increased dosage of SGD1 partially suppresses the osmosensitivity of pbs2 delta and hog1 delta mutants that are defective in the osmosignaling high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. The rescued mutants display a partially re-established transcriptional control of the osmostress-induced expression of GPD1, a target gene of the HOG pathway encoding NAD(+)-dependent glycerol 3-phosphate dehydrogenase, and a partially recovered hyperosmolarity-induced production of glycerol. Consistent with Sgd1p affecting the transcriptional control of GPD1, a functional green fluorescent protein tagged Sgd1p is localized to the cell nucleus.

DAPD (Emory University/Triangle Pharmaceuticals/Abbott Laboratories).

Triangle Pharmaceuticals is developing DAPD, a prodrug of the viral replication inhibitor dioxolane guanosine, as a potential therapy for HIV and HBV infection. Phase I/II dose range studies have commenced for HIV, and clinical development for HBV was to have commenced by late 1999 [319145], [319956]. Phase II trials are scheduled for the second quarter of 2001. The FDA has designated DAPD as a Fast Track product [365894]. DAPD is from a different nucleoside series to FTC and CS-92, which are also in development by Triangle. The compound may offer advantages over several nucleosides from other series that are already on the market because of its unique structure and pharmacological properties [247083]. Both DAPD and DXG are dioxolane purine nucleoside analogs [319660]. Preclinical data suggest DAPD may be of use in combination therapies for HIV-infected patients who are therapy-naive, in addition to patients who have previously received treatment and including those infected with drug-resistant strains of HIV-1 [341145], [341335]. Triangle licensed DAPD from Emory University [216900]. In June 1999, Triangle and Abbott Laboratories entered into an alliance for the development and marketing of six antiviral products, including DAPD [326824].

Defining functional drug-interaction domains on topoisomerase II by exploiting mechanistic differences between drug classes.

Topoisomerase II is the primary cellular target for a variety of antineoplastic drugs that are active against human cancers. These drugs exert their cytotoxic effects by stabilizing covalent topoisomerase II-cleaved DNA complexes that are fleeting intermediates in the catalytic cycle of the enzyme. Despite this common feature of drug action, a number of mechanistic differences between drug classes have been described. These mechanistic differences (including effects on DNA cleavage/religation, DNA strand passage, and adenosine triphosphate hydrolysis) were used as the basis for a series of competition experiments to determine whether different compounds share a common site of action on topoisomerase II or interact at distinct sites. Results of the present study strongly suggest that at least four structurally disparate antineoplastic drugs, etoposide, amsacrine, genistein, and the quinolone CP-115,953, share an overlapping interaction domain on the enzyme.

Nuclear transport mechanisms.

The term nuclear transport, refers to the movement of a large variety of macromolecules both into and out of the nucleus. Transport must be extremely selective, yet also very efficient. A single type of channel, the nuclear pore complex, mediates all movement across the nuclear envelope. Selectivity is achieved through the use of families of soluble factors that target substrates for import and export and deliver them to their appropriate intracellular destinations. We now have a fairly detailed understanding of the basic mechanisms of protein import into the nucleus. Many of these same principles can be applied to protein export and perhaps RNA export. This review will summarize the current status of what is known about various transport pathways and highlight the questions that remain to be answered.

The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo.

The small protein p10/Ntf2p has been implicated in protein import in vitro (Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10212-10216; Paschal, B. M., and Gerace, L.(1995) J. Cell Biol. 129, 925-937). Here we present the first evidence that demonstrates an essential in vivo role for the NTF2 gene product in nuclear transport. The NTF2 locus was identified in a screen for temperature-sensitive Saccharomyces cerevisiae mutants defective in the localization of nuclear proteins. Genetic analysis demonstrates that the NTF2 gene is essential for viability in budding yeast. Two temperature-sensitive mutants, ntf2-1 and ntf2-2, that each contain single point mutations in highly conserved amino acid residues show defects in the localization of nuclear proteins but not in the export of poly(A)+ RNA following a shift to the nonpermissive temperature. An epitope-tagged version of Ntf2p was used to show that the protein is concentrated at the nuclear envelope. Finally, the human gene under the control of the yeast promoter fully substitutes for the deleted yeast gene. Taken together, these results demonstrate the exquisite functional conservation of this protein throughout evolution and indicate a critical in vivo role in nuclear transport.