title: Development of a sensitive amplified luminescent proximity homogeneous assay to monitor the interactions between pTEFb and Tat.
abstract: The viral transactivator protein (Tat) plays an essential role in the replication of human immunodeficiency type 1 virus (HIV-1) by recruiting the host positive transcription elongation factor (pTEFb) to the RNA polymerase II transcription machinery to enable an efficient HIV-1 RNA elongation process. Blockade of the interaction between Tat and pTEFb represents a novel strategy for developing a new class of antiviral agents. In this study, we developed a homogeneous assay in AlphaLISA (amplified luminescent proximity homogeneous assay) format using His-tagged pTEFb and biotinylated Tat to monitor the interaction between Tat and pTEFb. On optimizing the assay conditions, the signal-to-background ratio was found to be greater than 10-fold. The assay was validated with untagged Tat and peptides known to compete with Tat for pTEFb binding. The Z' of the assay is greater than 0.5, indicating that the assay is robust and can be easily adapted to a high-throughput screening format. Furthermore, the affinity between Tat and pTEFb was determined to be approximately 20 pM, and only 7% of purified Tat was found to be active in forming tertiary complex with pTEFb. Development of this assay should facilitate the discovery of a new class of antiviral agents providing HIV-1 patients with broader treatment choices.
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Burlein
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Bahnck
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Triveni
authors.initials: T
authors.lastname: Bhatt
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Murphy
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Lemaire
authors.affiliation: Department of In Vitro Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Carroll
authors.affiliation: Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA. Electronic address: mingtain_lai@merck.com.
authors.forename: Ming-Tain
authors.initials: MT
authors.lastname: Lai
mesh_terms: D000818:Animals; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B
keywords: AlphaLISA; Assay development; HIV-1; Tat; pTEFb