CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine.

BACKGROUND: The 8-aminoquinoline (8AQ) drug primaquine (PQ) is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ's haemotoxic and anti-malarial properties are not fully understood. METHODS: In the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP) and mono-amine oxidase (MAO) families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements. RESULTS: Relative activity factor (RAF)-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species. CONCLUSIONS: As a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

Novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type 1 gene expression in primary macrophages.

CD8(+) T lymphocytes control human immunodeficiency virus type 1 (HIV-1) infection by a cytotoxic major histocompatibility complex-restricted pathway as well as by secretion of noncytotoxic soluble inhibitory factors. Several components of CD8(+) cell supernatants have been identified that contribute to the latter activity. In this study we report that prothymosin alpha (ProTalpha), a protein found in the cell culture medium of the herpesvirus saimiri-transformed CD8(+) T-cell line, K#1 50K, has potent HIV-1-inhibitory activity. Depletion of native ProTalpha from an HIV-1-inhibitory fraction of CD8(+) cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTalpha is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. In this report we demonstrate that ProTalpha suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTalpha protein potently inhibit HIV-1 long terminal repeat (LTR)-driven gene expression in macrophages. Furthermore studies using different promoters in lentiviral vectors (cytomegalovirus and phosphoglycerate kinase) revealed that suppression of viral replication by ProTalpha is not HIV LTR specific.

Identification of the surfactant protein A receptor 210 as the unconventional myosin 18A.

Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A.

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites.

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.

Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel.

We have described previously a cell surface channel that is highly selective for nucleic acids. Nucleic acid conductance is 10 pS and the channel is at least 10,000-fold more selective for oligodeoxynucleotides than any anion tested (1). Herein we provide evidence that the nucleic acid-conducting channel (NACh) is a heteromultimeric complex of at least two proteins; a 45-kDa pore-forming subunit (p45) and a 36-kDa regulatory subunit (p36). Reconstitution of p45 in planar lipid bilayers resulted in formation of a channel which gated in the absence of nucleic acid and which was more selective for anions (including oligonucleotide) than cations. This channel exhibited transitions from one level of current to another (or to the closed state); however the incidence of transitions was rare. Channel activity was not observed when p36 was reconstituted alone. Reconstitution of p36 with p45 restored nucleic acid dependence and selectivity to the channel. Protein sequence analysis identified p36 as cytosolic malate dehydrogenase (cMDH). Experiments were performed to prove that cMDH is a regulatory subunit of NACh. Selective activity was observed when p45 was reconstituted with pig heart cMDH but not with mitochondrial MDH. Both the enzyme substrate l-malate and antiserum raised against cMDH block NACh activity. These data demonstrate that a nucleic acid conducting channel is a complex of at least two proteins, p45 and cMDH. Furthermore, these data establish that cMDH confers nucleic acid selectivity of the channel.