Fluorescent HTS Assays for Methyltransferases in Neurodegenerative Diseases

描述(申请人提供)：甲基化是一种普遍存在的可逆的共价修饰，用于控制不同生物分子的功能，包括它们的活性、稳定性和定位。与磷酸化一样，甲基化被用来调节蛋白质的功能，但除此之外，许多小分子也受到甲基化的调节，包括激素、神经递质、外源物质和类脂。人类体内存在着50多种不同的甲基转移酶(MT)酶，毫不奇怪，它们正在成为一系列疾病的治疗靶点。由于甲基化在调节神经递质功能中起着核心作用，它们在神经退行性疾病途径中的参与具有特殊的相关性。开发高选择性的MT调节剂显然是医学上的当务之急。然而，缺乏适用于高通量筛选(HTS)的稳健、灵活的酶分析方法，阻碍了实现这一目标的努力。为了满足这一需求，贝尔布鲁克实验室在第一阶段开发了一种新的荧光偏振免疫分析方法，用于检测所有MT反应的不变产物--S-同型半胱氨酸。这需要开发一种高度选择性的抗体，能够根据单个甲基区分SAH和底物S-腺苷蛋氨酸。Transcreener MT检测方法能够以均一的形式检测具有任何受体底物的任何MT，并且不使用偶联酶，从而消除了与替代方法相关的所有缺点。在第二阶段，我们建议1)迅速将基于第一阶段开发的多克隆抗体的第一代基于荧光偏振的Transcreener MT分析商业化，2)通过对重组IgG克隆进行定向突变来增加SAH抗体的选择性，从而增加MT分析的灵活性和易用性，3)通过将该分析方法格式化为第二种常用的荧光检测模式TRRET，从而增加HTS的市场渗透率，以及4)将新型MT分析与电子筛选相结合，开发五种小分子甲基转移酶的异构体选择性抑制剂。强大的通用荧光HTS分析方法和MT的选择性化学探针的可获得性将大大加快它们作为药物靶标的有效性，并进入制药药物发现流水线。

DESCRIPTION (provided by applicant): Methylation is a ubiquitous and reversible covalent modification used to control the function of diverse biomolecules including their activity, stability and localization. Like phosphorylation, methylation is used to modulate protein function, but in addition many small molecules are regulated by methylation including hormones, neurotransmitters, xenobiotics, and lipids. More than 50 distinct methyltransferase (MT's) enzymes are present in humans, and not surprisingly they are emerging as therapeutic targets for a broad range of diseases. Their involvement in neurodegenerative disease pathways is of special relevance because of the central role that methylation plays in regulating neurotransmitter function. The development of highly selective MT modulators is clearly a compelling medical priority. However, efforts to achieve this are being hampered by a lack of robust, flexible enzyme assay methods adaptable to high throughput screening (HTS). To address this need, in Phase I BellBrook Labs developed a novel fluorescence polarization immunoassay for S-adenosylhomocysteine (SAH), the invariant product of all MT reactions. This required development of a highly selective antibody that is able to differentiate between SAH and the substrate S-adenosylmethionine on the basis of a single methyl group. The Transcreener MT assay enables detection of any MT with any acceptor substrate in a homogenous format - and without the use of coupling enzymes - thus it eliminates all of the shortcomings associated with alternative methods. In Phase II we propose to 1) rapidly commercialize a first generation, fluorescence polarization-based Transcreener MT assay based of the polyclonal antibodies developed in Phase I, 2) increase flexibility and ease-of-use for the MT assay by using targeted mutagenesis of recombinant IgG clones to increase antibody selectivity for SAH, 3) increase HTS market penetration by formatting the assay for a second commonly used fluorescent detection mode, TR-FRET, and 4) use the novel MT assay combined with in silico screening to develop isoform selective inhibitors for five small molecule methyltransferases. The availability of robust, generic fluorescent HTS assay methods and selective chemical probes for MT's should greatly accelerate their validation as drug targets and movement into the pharma drug discovery pipeline.