Dental applications for a novel osteoclast inhibitor

描述（由申请人提供）：依诺沙星和Binhib16最近在体外实验中被确定为破骨细胞形成和功能的新型抑制剂。先前的研究表明，液泡H+ atp酶b亚基（v - atp酶）和肌动蛋白丝之间相互作用的抑制剂可能会阻断破骨细胞吸收骨的能力。基于计算机的小分子屏幕预测结合亚基B的肌动蛋白结合表面并抑制其与肌动蛋白丝的相互作用，确定了依诺沙星和Binhib16。这些分子随后在试管中被证明可以阻断纯兔肌动蛋白细丝与b亚基的结合。在骨化三醇刺激的小鼠骨髓培养中，研究了依诺沙星和Binhib16对破骨细胞形成和功能的影响。在浓度约为10 <M时，均可阻断破骨细胞的形成和预分化破骨细胞的功能。相反，根据碱性磷酸酶阳性细胞的数量判断，这些分子对骨髓培养中产生的成骨细胞数量或MC3T3-E1成骨细胞没有可检测到的影响。这些数据表明依诺沙星和Binhib16可能通过新的机制抑制破骨细胞，而对成骨细胞没有抑制作用。本提案的总体目标是进行初步测试，以确定依诺沙星和Binhib16是否有望作为治疗各种牙科应用的药物，在这些应用中抑制破骨细胞骨吸收将是有益的。提出以下中心假设：依诺沙星和Binhib16是一类新的治疗药物的第一个例子，通过靶向v - atp酶与肌动蛋白细胞骨架的结合来抑制破骨细胞骨吸收。提出了四个具体目标。在Aim 1中，我们将利用Elvax开发一种局部缓释依诺沙星和Binhib16的方法。许多研究已经将Elvax用于类似的应用程序。在第二项研究中，我们将在一个成熟的大鼠牙周炎模型中测试依诺沙星和Binhib16减少牙周骨质流失的能力。目的3将测试使用依诺沙星和Binhib16治疗是否会减少与利用已建立的雪貂系统重新植入相关的吸收。目的4将在大鼠模型中测试依诺沙星和Binhib16阻断正畸牙齿移动的能力。这可以提供一种提供正畸支抗的新方法。这项研究应该提供初步证据，关于v - atp酶-肌动蛋白相互作用抑制剂在牙科手术中的潜在应用。为了提供足够的初步数据来确定大规模临床前研究是否合理，对当前研究的支持是必要的。鉴于拟议的研究代表了一种将生物活性小分子用于牙科应用的新颖创新方法，并且可能产生巨大的临床影响，我们认为NIH以R21拨款的形式支持是有保证的。

DESCRIPTION (provided by applicant): Enoxacin and Binhib16 were recently identified as novel inhibitors of osteoclast formation and function in in vitro assays. Previous studies had indicated that inhibitors of an interaction between the B-subunit of vacuolar H+ATPase (V-ATPase) and actin filaments should block the ability of osteoclasts to resorb bone. A computer based screen for small molecules predicted to bind the actin binding surface of subunit B and inhibit its interaction with actin filaments identified enoxacin and Binhib16. These molecules were then shown to block binding of pure rabbit muscle actin filaments to B-subunit in the test tube. Enoxacin and Binhib16 were tested for effects on osteoclast formation and function in vitro in calcitriol-stimulated mouse marrow cultures. Both blocked osteoclast formation and the function of pre-differentiated osteoclasts at a concentration of approximately 10 <M. In contrast, these molecules did not have detectable effects on the number of osteoblasts produced in marrow cultures, as judged by the number of alkaline phosphatase positive cells, or on MC3T3-E1 osteoblasts. These data suggest that enoxacin and Binhib16 might inhibit osteoclasts by novel mechanisms, without having inhibitory effects on osteoblasts. The overall goal of this proposal is to perform preliminary tests to determine whether enoxacin and Binhib16 hold promise as agents for use in the treatment of various dental applications in which inhibition of osteoclastic bone resorption would be beneficial. The following central hypothesis is proposed: Enoxacin and Binhib16 are the first examples of a new class of therapeutic agents for the inhibition of osteoclastic bone resorption by targeting V-ATPase binding to the actin cytoskeleton. Four specific aims are suggested. In Aim 1, we will develop a method for sustained local release of enoxacin and Binhib16 making use of Elvax. Numerous studies have utilized Elvax for similar applications. In Aim 2, enoxacin and Binhib16 will be tested for its capacity to reduce periodontal bone loss in a well-established rat model of periodontitis. Aim 3 will test whether treatment with enoxacin and Binhib16 reduces resorption associated with re- implantation making use of an established ferret system. Aim 4 will test the capacity of enoxacin and Binhib16 to block orthodontic tooth movement in a rat model. This could provide a novel means for providing orthodontic anchorage. This study should provide initial evidence regarding the potential of inhibitors of V-ATPase-actin interactions for use in dental procedures. Support for the current studies is necessary in order to provide sufficient preliminary data to determine whether large scale pre-clinical studies are justified. Given that the proposed research represents a fresh and innovative approach to the use of bioactive small molecules for dental applications, and could have a large clinical impact, we believe that NIH support in the form of an R21 grant is warranted. PUBLIC HEALTH RELEVANCE: A bioactive agent that inhibits osteoclastic bone resorption would be useful for various dental applications. Novel small molecule inhibitors of osteoclast function, enoxacin and Binhib16, have been identified using a supercomputer-based assay. The current proposal will conduct pilot preclinical tests of these molecules in animal models to examine their suitability for periodontic, endodontic and orthodontic uses.