Development of Chemical Receptors for Proteins

本提案旨在利用可聚合的混合脂质体为蛋白质制造高选择性，健壮的化学受体。碳酸酐酶将用于初始优化研究；优化研究完成后，重点将转移到生物医学相关蛋白MMP-13上。基质金属蛋白酶（MMPs）是一类能够降解细胞外基质的含锌内肽酶。这些酶的过度表达与肿瘤的侵袭和转移有关。在这个家族中，胶原酶-3 （MMP-13）能够在中性ph下降解II型胶原。它在乳腺癌周围的成纤维细胞中被发现过表达。MMPs无处不在，是正常身体功能所必需的。由于一类特定酶的过度表达与疾病有关，因此选择性靶向一类酶对于治疗疾病而不破坏其他身体功能至关重要。具体目标总结如下。新型两性离子、阳离子和氢键可聚合脂类的合成合成新的可聚合脂质，能够络合过渡金属离子（如Cu2+， Zn2+等）或具有高亲和力（K大于10′5 M-1）的发光镧系金属离子（如Tb3+或Eu3+）。通过在脂质体上创建金属离子、离子对和氢键位点的三维模式，与MMP-13展示的模式互补，制造稳定的蛋白质化学受体。为了实现这一目标，混合可聚合脂质体将由离子脂质体、金属螯合脂质体和在其头基上具有伯胺部分的脂质体制备。可聚合二酰基磷脂胆碱（两性离子）将被用作这些脂质体的主要成分。制作完成后，这些脂质体（处于未聚合状态，高于凝胶转变温度）将被允许与蛋白质相互作用。脂质体表面的金属离子取向与MMP-13表面暴露的组氨酸模式互补。脂质体上的季铵头基将被蛋白质表面的酸性氨基酸残基（Asp, Glu）定位。能够形成氢键的蛋白质氨基酸侧链（例如，Ser, Thr, Lys, Asn， Gln和Arg）将与脂质体上的伯胺部分相互作用。这个平衡步骤的结果是在脂质体上形成一种金属离子、电荷和氢键位点的模式，与模板蛋白所展示的表面模式互补。脂质体上的图案将通过光聚合被锁定。聚合脂质体将通过一系列三维同步和互补的相互作用“识别”MMP-13；因此，结合将是强的和选择性的。发光光谱和微量热法测定结合亲和力和选择性利用电子还原光谱（EPR）、透射电镜（TEM）和原子力显微镜（AFM）对其结构进行表征。测试合成受体在转移性癌症检测、成像和抗癌药物递送方面的可能应用。

This proposal aims to fabricate highly selective, robust, chemical receptors for the proteins employing polymerizable mixed liposomes. Carbonic anhydrase will be used for the initial optimization studies; after the optimization studies are complete, focus will be shifted to a bio-medically relevant protein, MMP-13. Matrix metallo-proteinases (MMPs) are a class of zinc-containing endo-peptidases capable of degrading extra-cellular matrix. Over-expressions of a variety of these enzymes have been implicated in tumor invasion and metastasis. Out of this family, Collagenase-3 (MMP-13) is capable of degrading type II collagen at neutral pH. It has been found to be over-expressed in fibroblastic cells surrounding brest carcinoma. MMPs are ubiquitous and are required for normal body functions. Since over-expression of a particular class of these enzymes is associated with diseases, selective targeting to one class of these enzymes is of paramount importance in treating the disease without disrupting other body functions. The specific goals are summarized below. Synthesis of new polymerizable zwitter-ionic, cationic and hydrogen bond forming lipids; synthesis of new polymerizable lipids capable of complexing either a transition metal ion (e.g., Cu2+, Zn2+ etc.) or a luminescent lanthanide metal ion (e.g., Tb3+ or Eu3+) with high affinity (K greater than 10'5 M-1). Fabrication of stable, chemical receptors for proteins by creating a three-dimensional pattern of metal-ions, ion-pairs and hydrogen bonding sites on liposomes, complimentary to the pattern exhibited by the MMP-13. In order to achieve this objective, mixed polymerizable liposomes will be prepared with ionic lipids, metal-chelating lipids and lipids with primary amine moiety on their headgroups. Polymerizable diacyl phosphocholine (zwitter ionic) will be used as the major constituent of these liposomes. After fabrication, these liposomes (in the unpolymerized state, above the gel-transition temperature) will be allowed to interact with the protein. The metal ions on liposome surface will orient complementary to the pattern of surface-exposed histidines of MMP-13. Quaternary ammonium headgroups on the liposomes will be positioned by acidic amino acid residues (Asp, Glu) on the protein surface. Amino acid side chains of the protein capable of forming hydrogen bonds (e.g., Ser, Thr, Lys, Asn, Gln and Arg) will interact with the primary amine moieties on the liposome. The result of this equilibration step is the creation of a pattern of metal ions, charges and hydrogen bonding sites on the liposome complementary to the surface pattern exhibited by the template protein. The pattern on the liposome will then be locked by photo-polymerization. The polymerized liposomes will "recognize" MMP-13 by an array of simultaneous and complementary interactions in three dimensions; thus the binding will be strong and selective. Measurement of binding affinity and selectivity by luminescence spectroscopy and micro-calorimetry; structural characterization studies by EPR spectroscopy, transmission microscopy (TEM) and atomic force microscopy (AFM). Testing of the synthetic receptors for possible applications in detection of metastatic cancer, imaging and delivery of anti- cancer drugs.