Isozyme Selectivity among Triple Helix Cleaving Metalloproteinases

描述（由申请人提供）：几种基质金属蛋白酶（MMPs）可切割胶原原纤维中普遍存在的三螺旋肽，并且一些此类同工酶（“三重解旋酶”）的过度表达/激活可导致致病性疾病，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角度来看，检测和抑制这些酶具有重要意义。该研究将采用我们最近开发的基于脂质体的方法，对导致各种人类疾病的“三重解旋酶”进行同工酶选择性检测和脱敏/抑制。这些目标将通过配制两种类型的脂质体来实现：(1)对于同工酶选择性检测，我们将在脂质体表面配制三螺旋肽，在管腔中配制信号放大探针，这样三螺旋肽的酶切会使脂质体不稳定并释放信号产生探针，以促进对母体酶的检测。(2)对于同工酶选择性脱敏/抑制，我们将制定具有“活性位点”和“表面仿射”残基的脂质体（类似于我们的“双头”抑制剂设计方法），这样脂质体的脂质流动性将促进上述残基并置到同工酶的互补区域，以选择性地降低酶的活性。为了深入了解同工酶选择性实现上述目标的分子基础，以及微调我们的整体检测和脱敏方案，我们将描述不同脂偶联物在脂质体稳定/不稳定中的“头-头”和“尾-尾”相互作用的能量贡献，获得脂质体头部基团和三重解旋酶同工酶之间的表面互补性。并释放脂质体封装的探针作为信号放大系统。本研究的具体目标如下：(1)开发基于脂质体的信号扩增策略，用于三解旋酶的同工酶特异性检测：我们将制备脂质体，在表面含有同工酶选择性三螺旋肽，在管腔中含有小DNA片段。根据单肽单元的氨基酸序列、脂质体表面生成的三螺旋肽的紧密性以及脂质体与酶之间的静电表面互补性，单个三螺旋酶同工酶将选择性地识别和切割其同源的三螺旋肽（驻留在脂质体表面），破坏脂质体组装并释放其封装的DNA片段。后者将用作信号扩增探针，并将通过实时PCR方法进行检测。除了稳健、敏感和同工酶选择性外，我们基于脂质体的方法将选择性地和独特地检测三解旋酶同工酶的“催化活性”形式，而不是传统ELISA方法检测的总同工酶。(2)模拟脂质体表面作为三解旋酶同工酶选择性抑制剂的“双头”配体设计策略：我们将模拟我们的“双头”配体设计策略，将“活性位点”和“酶表面”的定向结合基团结合在脂质体表面，选择性地抑制一种三解旋酶同工酶，而不是另一种。脂质体表面的活性位点定向配体（一种羟酸盐抑制剂）将作为“诱饵”，以“锚定复合物”的形式引诱酶结合。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）并置到酶的位置。一旦三螺旋同工酶和脂质体组装体之间的稳定复合物形成，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，该脂质体将选择性地抑制其母三螺旋同工酶，而不是其他三螺旋同工酶

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.