Isozyme Selectivity among Triple Helix Cleaving Metalloproteinases

描述(申请人提供)：几种基质金属蛋白酶(MMPs)裂解三螺旋多肽，普遍存在于胶原纤维中，一些这种同工酶(“三螺旋酶”)的过度表达/激活会导致诸如关节炎、动脉粥样硬化、慢性阻塞性肺疾病、溃疡和各种癌症等致病条件。因此，从预防和/或治疗相关人类疾病的角度来看，对这些酶的检测和抑制具有重要意义。这项拟议的研究将使用我们最近开发的基于脂质体的方法来选择性地检测和脱敏/抑制导致各种人类疾病的三重螺旋酶。这些目标将通过配制两种类型的脂质体来实现：(1)对于同工酶的选择性检测，我们将在表面形成三螺旋多肽的脂质体，在管腔内形成信号放大探针，这样三螺旋多肽的酶促裂解会破坏脂质体的稳定性，并释放它们的信号产生探针，从而促进亲本酶的检测。(2)对于同工酶的选择性脱敏/抑制，我们将形成具有“活性部位”和“表面仿射”残基的脂质体(类似于我们的“双管齐下”的抑制剂设计方法)，使脂质体的脂运动有助于将上述残基并列到同工酶的互补区域，从而选择性地降低酶的活性。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，我们将描述不同脂联物之间的“头-头”和“尾-尾”相互作用在稳定/不稳定脂质体中的能量贡献，获得脂质体头部基团和三重螺旋酶同工酶之间的表面互补，以及脂质体包裹的探针的释放作为信号放大系统。本研究的具体目的如下：(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：我们将制备脂质体，在表面含有同工酶选择性的三螺旋多肽，在管腔中含有一小段DNA片段。根据单体多肽单元的氨基酸序列、生成的三螺旋多肽在脂质体表面的致密性以及脂质体和酶之间的静电表面互补性，个别的三重螺旋酶同工酶将选择性地识别和切割它们的同源三螺旋多肽(位于脂质体表面)，破坏脂质体组装并释放它们包裹的DNA片段。后者将被用作信号放大探针，并将通过实时荧光聚合酶链式反应方法进行检测。除了稳健、灵敏和同工酶选择性外，我们基于脂质体的方法将选择性和唯一地检测三重螺旋酶同工酶的“催化活性”形式，而不是传统的ELISA法检测的总同工酶。(2)模拟脂质体表面的“双叉”配基设计策略，作为三重螺旋酶的同工酶选择性抑制剂：我们将模拟我们的“双叉”配基设计策略，在脂质体表面加入“活性部位”和“酶的表面”定向结合基团，选择性地抑制一个三重螺旋酶同工酶。脂质体表面的活性部位导向配体(羟甲酸类抑制剂)将作为一个“诱饵”，以“锚定复合体”的形式吸引酶的结合。随后，脂质体的脂质流动性将促进互补的带电残基(从脂质体表面)并列到酶的位置。一旦三螺旋同工酶和脂质体组合物之间形成稳定的络合物，我们将锁定脂质体的流动性(通过光聚合或双功能交联法)，以生成“模板化”脂质体，它将选择性地抑制其亲本三螺旋同工酶，而不是另一个三元。 解旋酶同工酶。(3)确定“头-头”和“尾-尾”相互作用在调节脂质体-三螺旋酶相互作用中的能量贡献：通过在“头”区表面暴露的三螺旋多肽和在“头”和“尾”区的荧光探针制备脂质体，我们将研究“头-头”和“尾-尾”相互作用对三螺旋酶与脂质体结合的能量贡献及其通过裂解三螺旋多肽而使其失稳。从这些研究中获得的机理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方案。这些目标将通过使用合成有机化学、分子生物学、电子光谱学、动力学和热力学技术来实现。这项“基础”“基础”研究的结果将被应用于个体三重螺旋酶同工酶的检测和脱敏/抑制，用于相关人类疾病的治疗干预。

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.