Fluorescent HTS Assays for Methyltransferases in Neurodegenerative Diseases

描述（由申请人提供）：甲基化是一种普遍存在的、可逆的共价修饰，用于控制多种生物分子的功能，包括活性、稳定性和定位。像磷酸化一样，甲基化也用于调节蛋白质功能，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、异种生物和脂质。人类体内存在超过50种不同的甲基转移酶（MT’s），毫不奇怪，它们正成为广泛疾病的治疗靶点。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通路中的参与具有特殊的相关性。高选择性MT调节剂的发展显然是一个引人注目的医疗优先事项。然而，由于缺乏适用于高通量筛选（HTS）的稳健、灵活的酶分析方法，实现这一目标的努力受到阻碍。为了满足这一需求，BellBrook实验室在第一阶段开发了一种新的荧光偏振免疫测定方法，用于检测所有MT反应的不变产物s -腺苷型同型半胱氨酸（SAH）。这需要开发一种高选择性抗体，能够根据单个甲基区分SAH和底物s -腺苷蛋氨酸。Transcreener MT检测能够以均质形式检测具有任何受体底物的任何MT，并且无需使用偶联酶，因此它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)快速商业化第一代基于荧光偏振的Transcreener MT检测，该检测基于第一阶段开发的多克隆抗体；2)通过对重组IgG克隆进行靶向诱变，以增加SAH抗体的选择性，从而增加MT检测的灵活性和易用性；3)通过将该检测设置为第二种常用的荧光检测模式（TR-FRET），增加HTS的市场渗透率。4)利用新型MT检测与硅筛选相结合，开发5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检测方法和选择性MT化学探针的可用性将大大加快其作为药物靶点的验证，并进入制药药物发现管道。

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.