Development of antibacterial agents and materials

目的1：研究材料成分如何决定机械性能。我们最初的研究集中在由聚葡聚糖醛（PDA）和模型含胺蛋白、牛血清白蛋白（BSA）以及聚乙烯亚胺（PEI）（一种高含量的胺聚合物）形成的粘合剂上。我们系统地研究了PDA链长、醛含量和醛/胺（CHO/NH2）网络比对材料形成速度、内聚性和粘合性能的影响。我们发现，这些参数都可以改变，以制备生物胶粘剂，其存储模量范围为102-105 Pa，胶粘剂强度范围为1-6 kPa，符合临床胶粘剂的要求。此外，通过改变（CHO/NH2）的比例，材料的形成速度可以从几秒调整到几分钟。此外，退化可以从几天调整到几年。目的2：研究材料的形成和水解机理。在体外和体内对含有eGFP和IL-2（一种重要的蛋白质免疫调节剂）的pda黏合剂的降解研究表明，除了纯蛋白外，还释放了被葡聚糖永久连接片段高度功能化的蛋白。如果蛋白质按照预期通过纯亚胺化学交联到PDA网络中，那么基于水解的降解应该只释放天然蛋白质。分析研究表明，除了生成亚胺外，还发生了随时间变化的美拉德反应。反应开始于亚胺交联，接着是Amadori重排，产生非水解酮胺，酮胺进一步反应，产生复杂的美拉德产物混合物。目标3：开发临床应用的粘接剂。多柔比星（DOX）的递送：我们通过混合DOX， PDA和BSA制备了一种多组分粘合剂。胶粘剂具有机械刚性（10kPa），能很好地粘附于组织（胶粘剂应力4kPa）。这种DOX- pda - bsa粘合剂缓慢水解（2个月），局部释放游离DOX、DOX- pda和由这三种成分组成的11nm颗粒，进入细胞，定位于细胞质，杀死A549肺癌细胞。确定细胞对SVS-1的抗性机制。我们进行了一项系统的调查，以确定癌细胞是否会产生耐药性。我们的报告首次表明真核细胞确实可以对acp产生耐药性。在与Dr. Klar （CCR）的合作中，我们首先利用S. pombe裂变酵母快速鉴定了三种不同的功能丧失基因突变，这些突变赋予了对SVS-1的抗性。酵母的遗传和机制研究促进了在哺乳动物癌细胞中类似机制的发现。有趣的是，这两种生物都是通过改变其细胞表面聚糖而不是直接改变其脂质膜的组成而产生耐药性的，而脂质膜是ACP作用的目标。糖基化的变化（酵母的丙酮酰化半乳糖和人类癌细胞的唾液酸）导致细胞表面静电电荷的减少。这反过来又减少了多肽在细胞表面积聚的潜力，并赋予了抵抗力。鉴于使用模型膜研究acp的大量研究，我们的工作表明聚糖的作用可能被低估了。目的2：调节SVS-1的活性。SVS-1与细胞相互作用的机制是复杂的，不仅取决于肽的结构特征，还取决于呈递给细胞的肽的浓度。当SVS-1浓度低于其溶解IC50 （5um）时，该肽与膜结合，但不溶解。相反，它会迅速进入细胞，进入细胞质，并随着时间的推移进入细胞核。因此，SVS-1既是一种裂解性ACP，也是一种细胞穿透肽（CPP），取决于浓度。作为一种CPP， SVS-1通过膜的直接易位和粘连蛋白依赖的内吞作用进入细胞。虽然我们利用了sbs -1作为CPP，但由于其溶解和细胞穿透活性的浓度窗口窄，一些递送应用变得复杂。此外，如果SVS-1完全通过直接易位进入细胞，从而避免了内体逃逸的问题，这将是最理想的。通过分子设计分离和分离膜干扰肽的活性和细胞进入机制是极具挑战性的。然而，在研究SVS-1的过程中，我们发现肽的折叠倾向与其裂解活性和内吞摄取倾向之间存在相关关系；SVS-1的结构衍生物显示出更高的折叠倾向，更容易裂解为acp，更容易被内吞为cpp。基于这一观察，我们假设如果可以设计一种内在无序的膜活性肽，它应该避免内吞作用，通过直接易位进入细胞，并且细胞毒性较小。我们设计了一种肽，CLIP6，它在其序列中含有一个关键的谷氨酸残基，该残基破坏了肽的两亲性，使其无法折叠。我们还发现，CLIP6本质上是紊乱的，并且只通过非内体机制进入细胞，同时具有显著的细胞相容性和血清稳定性。此外，CLIP6可以将膜不透货物直接运送到细胞的细胞质中。目标3：开发SVS-1及其衍生物的治疗应用。小分子传递：在药物发现活动中发现的许多铅分子由于溶解度差和细胞渗透性低而被排除在考虑之外。这些孤立分子如果被适当地溶解和输送，可能具有临床价值。在对SVS-1效用的初步评估中，模型疏水药物紫杉醇（PTX）通过自焚连接物连接，其溶解度提高了1000倍。在体外和体内，SVS-1成功地将PTX传递并释放到癌细胞中，在异种移植小鼠模型中，肿瘤负荷显著降低。蛋白质递送：生物制剂在临床上产生了巨大的影响，尽管它们目前只能处理细胞表面的目标。将生物制剂输送到细胞中的能力将大大增加它们的效用。CPPs，如TAT，已被用于递送生物制剂，但几乎所有CPPs都通过一定程度的内吞作用进入细胞，限制了它们的有效性。我们基于SVS-1和CLIP6开发了一个具有表达能力的细胞穿透肽（XCPs）家族，该家族用包含所有l-残基的序列取代了它们的d -脯氨酸转变。XCPs可以直接重组地与蛋白质融合，从而消除了化学连接的需要。我们目前正在优化我们的第一代设计，它显示了直接转运和内吞摄取机制之间的平等分配。

Aim 1: Investigate how material composition dictates mechanical properties. Our initial studies centered on adhesives formed by polydextran aldehyde (PDA) and the model amine-containing protein, bovine serum albumin (BSA), as well as polyethylenimine (PEI), a high-content amine polymer. We systematically studied how PDA chain length, aldehyde content, and the aldehyde/amine (CHO/NH2) network ratio influenced the rate of material formation, cohesive, and adhesive properties of resultant materials. We found that each of these parameters can be varied to prepare bioadhesives that range in storage moduli from 102-105 Pa and adhesive strengths from 1-6 kPa, which is on the order of clinical adhesives. In addition, the rate of material formation can be tuned from seconds to minutes after delivery by varying the (CHO/NH2) ratio. Further, degradation can be tuned from days-to-years. Aim 2: Investigate mechanism of material formation and hydrolysis. In vitro and in vivo degradation studies of PDA-based adhesives employing eGFP and IL-2, an important protein immunoregulator, indicated that along with pure protein, proteins heavily functionalized with permanently ligated fragments of dextran were also released. If protein had been crosslinked into the PDA network via pure imine chemistry as intended, then hydrolysis-based degradation should have released only native protein. Analytical studies revealed that in addition to imine formation, a time-dependent Maillard reaction was occurring. The reaction begins with imine crosslinking, followed by an Amadori rearrangement affording non-hydrolyzing keto amines that react further affording a complex mixture of Maillard products. Aim 3: Develop adhesives towards clinical applications. Delivery of doxorubicin (DOX): We prepared a multicomponent adhesive by mixing DOX, PDA, and BSA. The adhesive is mechanically rigid (10kPa) and adheres avidly to tissue (adhesive stress 4kPa). This DOX-PDA-BSA adhesive slowly hydrolyzes (2 months), locally releasing free DOX, DOX-PDA and 11nm particles composed of all three components that enter cells, localize to the cytoplasm and kill A549 lung carcinoma cells With respect to the cell penetrating peptides: Aim 1. Determine cellular mechanisms of resistance towards SVS-1. We undertook a systematic investigation to determine if cancer cells can develop resistance. Our report is the first to show that eukaryotic cells can, indeed, develop resistance to ACPs. In collaboration with Dr. Klar (CCR), we first utilized S. pombe fission yeast to rapidly identify three different loss-of-function gene mutations that conferred resistance to SVS-1. Genetic and mechanistic studies in yeast facilitated discovery of similar mechanisms operating in mammalian cancer cells. Interestingly, both organisms developed resistance by altering their cell-surface glycans rather than directly changing the composition of their lipid membranes, the target of ACP action. Changes in glycosylation (pyruvylated galactose for yeast and sialic acid for human cancer cells) led to a reduction in the electrostatic charge at the cell surface. This, in turn, reduces the potential of peptide to accumulate at the surface of cells and confers resistance. Given the plethora of studies using model membranes to study ACPs, our work shows that the role of glycans may have been underappreciated. Aim 2: Modulating the activity of SVS-1. The mechanisms by which SVS-1 interacts with cells are complex and dependent not only on the peptide's structural features, but also on the concentration of peptide presented to cells. When cells are presented with concentrations of SVS-1 that are below its lytic IC50 ( 5 uM), the peptide engages the membrane, but is not lytic. Rather, it rapidly enters cells, accessing the cytoplasm and over time, the nucleus. Thus, SVS-1 is both a lytic ACP and a cell penetrating peptide (CPP), depending on concentration. As a CPP, SVS-1 enters cells by both direct translocation through the membrane and clatherin-dependent endocytosis. Although we exploit SVS-1 as a CPP, some delivery applications are complicated by the narrow concentration window differentiating its lytic and cell penetrating activities. Further, it would be optimal if SVS-1 entered cells purely by direct translocation, thus avoiding issues of endosomal escape. Decoupling and isolating the activity and cellular entry mechanisms of membrane-perturbing peptides by molecular design is extremely challenging. However, over the course of studying SVS-1, we discovered a correlative relationship between the peptide's folding propensity and both its lytic activity and its propensity for endocytic uptake; structural derivatives of SVS-1 that show a higher propensity to fold are more lytic as ACPs and are endocytosed more readily as CPPs. Based on this observation, we hypothesized that if an intrinsically disordered membrane-active peptide could be designed, it should avoid endocytosis, enter cells via direct translocation, and be less cytotoxic. We designed a peptide, CLIP6, which contains a key glutamate residue in its sequence that disrupts the amphiphilicity of the peptide rendering it incapable of folding. We also showed that CLIP6 is intrinsically disordered and exclusively enters cells by non-endosomal mechanisms, while being remarkably cytocompatible and serum-stable. Further, CLIP6 can deliver membrane-impermeable cargo directly to the cytoplasm of cells. Aim 3: Exploiting SVS-1 and its derivatives towards therapeutic applications. Small molecule delivery: Many lead molecules identified in drug discovery campaigns are eliminated from consideration due to poor solubility and low cell permeability. These orphaned molecules could have clinical value if solubilized and delivered properly. In an initial assessment of SVS-1's utility, the model hydrophobic drug Paclitaxel (PTX) was ligated via a self-immolative linker, increasing its solubility by 1000-fold. SVS-1 successfully delivered and released PTX to cancer cells in vitro and in vivo, where tumor burden was significantly reduced in a xenograft mouse model. Protein delivery: Biologics have made tremendous impact clinically despite the fact they can currently address only cell surface targets. The ability to deliver biologics into cells would vastly increase their utility. CPPs, such as TAT, have been used to deliver biologics, but nearly all enter cells through some degree of endocytosis, limiting their effectiveness. We developed a family of expression-capable cell-penetrating peptides (XCPs) based on SVS-1 and CLIP6, which replace their D-proline turn with sequences containing all L-residues. XCPs can be directly fused to proteins recombinantly, eliminating the need for chemical ligation. We are currently optimizing our first-generation designs, which show equal partitioning between direct translocation and endocytic uptake mechanisms.