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共价修饰，用于控制各种生物分子的功能，包括其活性、稳定性和定位。像磷酸化一样，甲基化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、外源性物质和脂质。超过50种不同的甲基转移酶（MT）存在于人类中，并且毫不奇怪，它们正在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发挥的核心作用，它们参与神经退行性疾病途径具有特殊意义。高选择性MT调节剂的开发显然是一个引人注目的医疗优先事项。然而，实现这一目标的努力受到缺乏适用于高通量筛选（HTS）的稳健、灵活的酶测定方法的阻碍。为了满足这一需求，在第一阶段，贝尔布鲁克实验室开发了一种新的S-腺苷高半胱氨酸（SAH），所有MT反应的不变产物的荧光偏振免疫测定。这需要开发一种高选择性抗体，该抗体能够基于单个甲基区分SAH和底物S-腺苷甲硫氨酸。Transcreener MT检测试剂盒能够以均质形式检测任何受体底物的任何MT-无需使用偶联酶-因此消除了与替代方法相关的所有缺点。在II期，我们提出1）基于I期开发的多克隆抗体的第一代基于荧光偏振的Transcreener MT测定快速商业化，2）通过使用重组IgG克隆的靶向诱变来增加MT测定的灵活性和易用性，以增加SAH的抗体选择性，3）通过格式化用于第二常用荧光检测模式TR-FRET的测定来增加HTS市场渗透，和4）使用新的MT测定结合计算机筛选来开发五种小分子甲基转移酶的异构体选择性抑制剂。稳健的通用荧光HTS测定方法和MT的选择性化学探针的可用性应大大加速其作为药物靶标的验证和进入制药药物发现管道的移动。

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.