Isozyme Selectivity among Triple Helix Cleaving Metalloproteinases

描述（由申请人提供）：几种基质金属蛋白酶（MMP）切割胶原纤维中普遍存在的三螺旋肽，并且一些这样的同工酶（“三螺旋酶”）的过表达/活化引起致病性病症，例如关节炎、动脉粥样硬化、慢性阻塞性肺病、溃疡和各种癌症等。因此，从预防和/或治疗相关人类疾病的观点来看，这些酶的检测和抑制是非常重要的。拟议的研究将采用我们最近开发的基于脂质体的同工酶选择性检测和脱敏/抑制“三重解旋酶”的方法，负责引起各种人类疾病。这些目的将通过配制两种类型的脂质体来实现：（1）对于同工酶选择性检测，我们将配制在表面上具有三螺旋肽并且在内腔中具有信号放大探针的脂质体，使得三螺旋肽的酶促切割将使脂质体不稳定并且释放它们的信号产生探针以促进亲本酶的检测。(2)对于同工酶选择性脱敏/抑制，我们将配制具有“活性位点”和“表面仿射”残基的脂质体（类似于我们的“双管齐下”抑制剂设计方法），使得脂质体的脂质移动将促进上述残基与同工酶的互补区域的并置，以选择性地敲低酶活性。为了深入了解实现上述目标的同工酶选择性的分子基础，以及微调我们的整体检测和脱敏方案，我们将描绘不同脂质缀合物在脂质体稳定/去稳定化中的“头-头”和“尾-尾”相互作用的积极贡献，实现脂质体头部基团和三重解旋酶同工酶之间的表面互补，以及释放脂质体包封的探针以用作信号放大系统。具体研究内容如下：（1）建立基于脂质体的三重螺旋酶同工酶特异性检测的信号放大策略：制备脂质体，其表面含有同工酶选择性三重螺旋肽，内腔含有小片段DNA。根据单体肽单元的氨基酸序列、脂质体表面上所得三螺旋肽的紧密性以及脂质体和酶之间的静电表面互补性，各个三螺旋解旋酶同工酶将选择性地识别并切割其同源三螺旋肽（驻留在脂质体表面上），使脂质体组装体不稳定并释放其包封的DNA片段。后者将用作信号放大探针，并将通过真实的时间PCR方法进行检测。除了是稳健的、灵敏的和同工酶选择性的之外，我们的基于脂质体的方法将选择性地和独特地检测三重解旋酶同工酶的“催化活性”形式，而不是如通过ELISA方法常规检测的总同工酶。(2)模拟脂质体表面上的“两叉”配体设计策略以作为针对三重解旋酶的同工酶选择性抑制剂：我们将通过在脂质体表面上并入“活性位点”和“酶表面”定向结合基团来模拟我们的“两叉”配体设计策略，以选择性地抑制一种三重解旋酶同工酶而不是另一种。脂质体表面上的活性位点定向配体（异羟肟酸盐抑制剂）将充当“诱饵”以“锚定复合物”的形式引诱酶的结合。随后，脂质体的脂质流动性将促进互补带电残基（从脂质体表面）与酶位点的并置。一旦三重螺旋同工酶和脂质体组装体之间形成稳定的复合物，我们将锁定脂质体脂质的流动性（通过光聚合或双功能交联）以产生“模板化”脂质体，其将选择性地抑制其亲本三重螺旋同工酶而不是其它三重螺旋同工酶。 解旋酶同工酶(3)确定“头-头”和“尾-尾”相互作用在调节脂质体-三重解旋酶相互作用中的能量贡献：通过配制在“头”区具有表面暴露的三螺旋肽且在“头”和“尾”区具有荧光探针的脂质体，我们将研究“头-头”和“尾-尾”的能量贡献，三重螺旋酶与脂质体的结合的相互作用和它们通过三重螺旋肽的切割的去稳定化。从这些研究中获得的机制见解将被用于微调基于脂质体的三重解旋酶同工酶的检测和脱敏协议。这些目标将通过采用合成有机化学、分子生物学、电子光谱学、动力学和热力学技术来实现。这一“基础”“基础”研究的结果将在检测和脱敏/抑制个体三重解旋酶同工酶对相关人类疾病的治疗干预中找到应用。

DESCRIPTION (provided by applicant): Several matrix metalloproteinases (MMPs) cleave triple helical peptides, prevalent in collagen fibrils, and the overexpression/activation of some such isozymes ("triple helicases") cause pathogenic conditions such as arthritis, atherosclerosis, chronic obstructive pulmonary disease, ulcers, and various cancers among others. Hence, the detection and inhibition of those enzymes are of significant importance from the point of view of prevention and/or treatment of associated human diseases. The proposed research will employ our recently developed liposome-based methodologies for the isozyme selective detection and desensitization/inhibition of "triple helicases", responsible for causing various human diseases. These objectives will be accomplished by formulating two-types of liposomes: (1) For isozyme selective detection, we will formulate liposomes with triple helical peptides on the surface and the signal amplification probes in the lumen, such that the enzymatic cleavage of the triple helical peptides would destabilize the liposomes and release their signal generating probes to facilitate the detection of the parent enzyme. (2) For isozyme selective desensitization/inhibition, we will formulate liposomes with "active site" and "surface affine" residues (akin to our "two-prong" inhibitor designing approach) such that the lipid mobilit of liposomes would facilitate juxtaposition of the above residues to the complementary regions of the isozymes to selectively knock down the enzyme activity. To gain insight into the molecular basis of isozyme selectivity in achieving the above objectives, as well as fine-tuning our overall detection and desensitization protocols, we will delineate the energetic contributions of "head-head" and "tail-tail" interactions among different lipo-conjugates in stabilization/destabilizationof liposomes, attaining surface complementarity between liposomal head groups and triple helicase isozymes, and the release of the liposome's encapsulated probes to serve as the signal amplification system. The Specific Aims of the proposed research are as follows: (1) Develop the liposome-based signal amplification strategy for the isozyme specific detection of triple helicases: We will formulate liposomes, harboring isozyme selective triple helical peptides on the surface and a small DNA fragment in the lumen. Depending on the amino acid sequences of the monomeric peptide units, compactness of resultant triple helical peptides on the liposomal surface, and the electrostatic surface complementarity between liposomes and the enzymes, individual triple helicase isozymes will selectively recognize and cleave their cognate triple helical peptides (resident on the liposomal surface), destabilize the liposomal assembly and release their encapsulated DNA fragment. The latter will be utilized as the signal amplification probe, and will be detected via the real time PCR method. Besides being robust, sensitive, and isozyme selective, our liposome based approach will selectively and uniquely detect the "catalytically active" forms of triple helicase isozymes, and not the total isozymes as conventionally detected by the ELISA method. (2) Simulate the "two-prong" ligand designing strategy on the liposome surface to serve as the isozyme selective inhibitors against triple helicases: We will simulate our "two-prong" ligand designing strategy by incorporating both "active site" and the "enzyme's surface" directed binding groups on the liposome surface for selectively inhibiting one triple helicase isozyme in preference to the other. The active site directed ligand (a hydroxamate inhibitor) on the liposome surface will serve as a "bait" to lure the binding of the enzyme in the form of an "anchored complex". Subsequently, the liposomal lipid mobility will facilitate juxtaposition of the complementary charged residues (from the liposomal surface) to the enzyme's site. Once the stable complex between a triple helical isozyme and the liposomal assembly is formed, we will lock the mobility of the liposomal lipids (via photo-polymerization or bifunctional cross- linking) to generate the "templated" liposomes, which will selectively inhibit its parent triple helicase isozyme in preference to the other triple helicase isozymes. (3) Ascertain the energetic contributions of "head-head" and "tail-tail" interaction in modulating the liposome-triple helicase interactions: By formulating liposomes with surface exposed triple helical peptides in the "head" region and fluorescence probes at both "head" and "tail" regions, we will investigate the energetic contribution of "head-head" and "tail-tail" interactions on binding of triple helicases to liposomes and their destabilization via the cleavage of the triple helical peptides. The mechanistic insights gained from these studies will be utilized toward fine-tuning the liposome based detection and desensitization protocols for triple helicase isozymes. These objectives will be accomplished by employing the techniques of synthetic organic chemistry, molecular biology, electronic spectroscopy, kinetics and thermodynamics. The outcome of this "basic" "fundamental" research will find applications in detection and desensitization/inhibition of individual triple helicase isozymes toward therapeutic intervention in associated human diseases.