Development of Chemical Receptors for Proteins

该提议旨在采用可聚合混合脂质体制造用于蛋白质的高选择性、稳健的化学受体。 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质MMP-13。基质金属蛋白酶（matrix metalloproteinases，MMPs）是一类能降解细胞外基质的含锌多肽内切酶。多种这些酶的过表达已经涉及肿瘤侵袭和转移。 胶原酶-3（MMP-13）在中性pH下能够降解II型胶原。已发现其在乳腺癌周围的成纤维细胞中过度表达。 MMP是普遍存在的，并且是正常身体功能所必需的。 由于这些酶的特定类别的过表达与疾病相关，因此选择性靶向这些酶的一类对于治疗疾病而不破坏其他身体功能至关重要。 具体目标概述如下。新的可聚合两性离子、阳离子和氢键形成脂质的合成;能够络合过渡金属离子（例如，Cu 2+、Zn 2+等）或发光镧系金属离子（例如，Tb ~（3+）或Eu ~（3+））。通过在脂质体上产生金属离子、离子对和氢键结合位点的三维图案来制造蛋白质的稳定化学受体，与MMP-13所表现出的图案互补。 为了实现这一目标，将用离子脂质、金属螯合脂质和在其头基上具有伯胺部分的脂质制备混合可聚合脂质体。 可聚合的二酰基磷酸胆碱（两性离子）将用作这些脂质体的主要成分。在制造之后，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。 脂质体表面上的金属离子将与MMP-13的表面暴露的组氨酸图案互补地取向。 脂质体上的季铵头基将通过蛋白质表面上的酸性氨基酸残基（Asp、Glu）定位。 能够形成氢键的蛋白质的氨基酸侧链（例如，Ser、Thr、Lys、Asn、Gln和Arg）将与脂质体上的伯胺部分相互作用。该平衡步骤的结果是在脂质体上产生金属离子、电荷和氢键合位点的图案，其与模板蛋白质所表现出的表面图案互补。 然后，脂质体上的图案将通过光聚合被锁定。 聚合的脂质体将通过一系列三维的同时和互补的相互作用“识别”MMP-13;因此结合将是强的和选择性的。通过发光光谱和微量热法测量结合亲和力和选择性;通过EPR光谱、透射显微镜（TEM）和原子力显微镜（AFM）进行结构表征研究。测试合成受体在转移性癌症检测、成像和抗癌药物递送中的可能应用。

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.