High-throughput profiling of proteases with phage and arrays

使用噬菌体和阵列对蛋白酶进行高通量分析

• 批准号: 10602245
• 负责人: BRIAN KENNETH KAY
• 金额: $ 25.68万
• 依托单位: TANGO BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10602245
• 关键词: Amino Acids; Antibodies; Apoptosis; Autophagocytosis; Bacteriophages; Binding; Biological Assay; Biological Sciences; C-terminal; Cell secretion; Chicago; Client; Clinical Trials; Cloning; Collaborations; Cysteine; Development; Disease; Drug Design; Enzymes; Escherichia coli; Eukaryotic Cell; Exclusion; Extracellular Matrix; Fluorescence; Frequencies; Future; Genes; Human; Incubated; Infection; Kanamycin Resistance; Knock-out; Libraries; Licensing; Location; Maps; Mass Spectrum Analysis; Metalloproteases; Methods; Modeling; Monitor; N-terminal; Neoplasm Metastasis; Nucleotides; Oligonucleotides; Output; Peptide Hydrolases; Peptide Library; Peptide Synthesis; Peptides; Phage Display; Phase; Play; Protease Inhibitor; Proteins; Proteolysis; Protocols documentation; Randomized; Recombinants; Role; Scanning; Services; Signal Transduction; Site; Small Business Innovation Research Grant; Small Molecule Chemical Library; Specificity; Spottings; Streptavidin; Substrate Specificity; Surface; Therapeutic; United States National Institutes of Health; Viral; Virion; Work; anti-cancer; cancer cell; cell growth; cellular targeting; combinatorial; covalent bond; density; drug discovery; experimental study; follow-up; homologous recombination; human disease; improved; inhibitor; innovation; magnetic beads; neutravidin; next generation sequencing; pathogen; peptidomimetics; phage vector; phosphoramidite; protein aminoacid sequence; resistance gene; screening; spatiotemporal; synthetic peptide; vector

Proteases represent an important class of enzymes in all eukaryotic cells where they play critical roles in activating and destroying proteins during cell growth, development, cell signaling, apoptosis, and autophagy. Misregulation, however, can lead to disease; for example, cancer cells often secrete metalloproteases that degrade the extracellular matrix, permitting cancers cells to metastasize and take root at various locations in the body. Proteases work by recogniz- ing linear regions, typically 6 to 8 residues, in their substrates and hydrolyzing a peptide bond, thereby fragmenting their substrate into N-terminal and C-terminal fragments. Understanding the range of peptide sequences cleaved by a protease, termed the "proteolysis signature" or "substrate profile", has many scientific implications and practical benefits. One powerful method for determining the proteolysis signature or substrate profile of a recombinant protease is through the use of combinatorial peptide libraries. In this effort, there are two specific aims. In Aim #1, we will develop an improved phage vector for protease site identification by allowing for more efficient cleavage of recognized sequences, more even representation of amino acids, and identification of longer recognition sequences (up to 10 AA).The vectors will carry the kanamycin resistance gene and a second copy of gene III that is tagged at the N-terminus with the AviTag and has the same cloning site cassette at three different locations. After the vectors have been demonstrated to be suitable as substrates for two model proteases, oligonucleotides will be ordered with a 10-mer region randomized with 19 trinucleotide phosphoramidites at equal frequency encoding 19 amino acids (excluding cysteine) for cloning into each site and large (> 1010) libraries created. The OmpT gene in the bacterial strain, XL1-Blue, will be knocked out by homologous recombination to eliminate the loss of phage-displayed peptides in the libraries that carry dibasic residues. In Aim #2, Tango will evaluate the utility of the new libraries with two commercially available proteases, the human metalloprotease, ADAM15, and the Escherichia coli protease, DegP. Virions will be captured on neutravidin-coated magnetic beads, released with each protease, and the output will be sequenced by Next Generation Sequencing (NGS). Nimble Therapeutics will then synthesize two million peptides on arrays, corresponding to 10 or more copies of each peptide sequence discovered by NGS, along with systematic amino acid replacements of any motifs identified among the selected peptides. The peptides will carry a peptide tag at their C-termini, which binds to fluorescent streptavidin, and a PEG linker at their N-termini to distance them it away from the array surface. Arrays will be incubated with the pro- tease, incubated with fluorescent streptavidin, and scans will reveal which spots lose fluores- cence due to proteolysis. To confirm that such peptides are true substrates for their respective protease, Tango will order 20 synthetic peptides for each and determine the cleavage site by mass spectrometry. Successful completion of the proposed experiments will create great value. Tango will build proprietary phage libraries and refine a standard operating protocol (SOP) with which it can determine the substrate profile for any client protease. A future Phase II SBIR appli- cation would enable Tango to define the profile 50 human and pathogen proteases, which would create licensing opportunities for the development of peptidomimetic inhibitors to treat human disease and infection.

蛋白酶在所有真核细胞中代表着重要的酶 在细胞生长，发育，细胞信号传导中激活和破坏蛋白质的关键作用 凋亡和自噬。然而，正直会导致疾病。例如，癌症 细胞经常分泌金属蛋白酶，使细胞外基质降解，允许癌症 细胞转移并扎根在体内的各个位置。蛋白酶通过识别来起作用 - 在其底物中，线性区域，通常为6至8个残基，并水解肽键， 从而将其底物分解为N末端和C末端片段。理解 蛋白酶裂解的肽序列的范围称为“蛋白水解特征”或 “底物概况”具有许多科学含义和实际好处。一种强大的方法 用于确定重组蛋白酶的蛋白水解特征或底物概况是 通过使用组合肽库。在这项工作中，有两个具体的目标。在 AIM＃1，我们将通过允许使用的蛋白酶站点识别的改进的噬菌体向量 公认序列的更有效切割，甚至更高的氨基酸表示， 并识别更长的识别序列（最多10个AA）。向量将携带 卡纳霉素的抗性基因和第二份基因III副本，该基因在N末端标记为 Avitag在三个不同位置具有相同的克隆位点盒式盒式盒式盒式盒式盒子。向量之后 已被证明是两个模型蛋白酶的底物，寡核苷酸 将用一个随机分组的10-摩尔区域与19个三核苷酸磷酰胺的区域相等 编码19个氨基酸（不包括半胱氨酸）的频率用于克隆每个位置和大型（> 1010）创建的库。细菌菌株中的OMPT基因XL1-蓝色将被淘汰 同源重组，以消除文库中噬菌体斑点肽的损失 携带二元残留物。在AIM＃2中，Tango将用两个评估新图书馆的实用性 市售的蛋白酶，人类金属蛋白酶，ADAM15和Escherichia 大肠杆菌蛋白酶，DEGP。病毒体将被捕获在中性素蛋白涂层的磁珠上，释放 使用每个蛋白酶，并且输出将通过下一代测序（NGS）进行测序。 然后，Nimble Therapeutics将在阵列上合成200万肽，对应于10或 NGS发现的每个肽序列的更多副本以及系统的氨基酸 替换所选肽之间确定的任何基序。肽将携带 在其C末端的肽标签，该肽与荧光链霉亲和素结合的肽标签，在其C-termini上结合的肽标签 N末端将其远离阵列表面。阵列将与Pro-一起孵化 取笑，与荧光链霉亲和素一起孵育，扫描将显示哪些斑点失去荧光 - 由于蛋白水解而引起的。确认这种肽是其各自的真正底物 蛋白酶，探戈将为每种订购20种合成肽，并通过 质谱法。成功完成拟议的实验将创造巨大的价值。 探戈将建立专有的噬菌体库，并与 它可以确定任何客户端蛋白酶的基板配置文件。未来的II期SBIR应用 阳离子将使探戈能够定义50个人类和病原体蛋白酶，这将 创造许可机会，以开发肽症抑制剂来治疗人类 疾病和感染。