Genecopoeia Inc, 9620 Medical Center Drive #101, 20850, Rockville, MD, USA.
Thromb J. 2013 Jan 30;11(1):2. doi: 10.1186/1477-9560-11-2.
Previous animal studies by us and others have indicated that catheter-administered plasmin or its des-kringle derivatives may be more appropriate alternatives to plasminogen activators for treating thrombolytic diseases, since it has a very short serum half-life and therefore does not result in hemorrhaging. We have previously produced recombinant miniPlasmin (mPlasmin) that was proven suitable for treating peripheral arterial occlusion in animal models. However, our previous results showed that non-specific cleavage at position K698 of mPlasmin during activation hindered the further development of this promising therapeutic candidate. In order to minimize or eliminate the non-specific cleavage problem, we performed saturation mutagenesis at the K698 position to develop a mutant form of mPlasmin for thrombolytic therapy.
We changed K698 to 16 other amino acids, with preferred E. coli codons. Each of these mutants were expressed in E. coli as inclusion bodies and then refolded, purified, and subsequently characterized by detailed kinetic assays/experiments/studies which identified highly active mutants devoid of non-specific cleavage.
Activation studies indicated that at those conditions in which the wild type enzyme is cut at the non-specific position K698, the active mutants can be activated without being cleaved at this position.
From the above results, we selected two mutants, K698Q and K698N, as our lead candidates for further thrombolytic drug developments. The selected mutants are potentially better therapeutic candidates for thrombolytic therapy.
我们和其他人之前的动物研究表明，与纤溶酶原激活剂相比，经导管给予纤溶酶或其去kringle 衍生物可能是更合适的溶栓疾病治疗替代方法，因为它的血清半衰期非常短，因此不会导致出血。我们之前已经生产出了重组 miniPlasmin（mPlasmin），它已被证明在动物模型中适用于治疗外周动脉闭塞。然而，我们之前的结果表明，在激活过程中 mPlasmin 在位置 K698 的非特异性切割阻碍了这一有前途的治疗候选药物的进一步发展。为了最小化或消除非特异性切割问题，我们在 K698 位置进行饱和突变，以开发用于溶栓治疗的 mPlasmin 突变体形式。
我们将 K698 替换为 16 个其他氨基酸，并使用优选的大肠杆菌密码子。这些突变体中的每一个都在大肠杆菌中作为包涵体表达，然后进行重折叠、纯化，并随后通过详细的动力学测定/实验/研究进行表征，这些研究确定了没有非特异性切割的高活性突变体。
激活研究表明，在野生型酶在非特异性位置 K698 被切割的那些条件下，活性突变体可以在不被切割的情况下被激活。
根据上述结果，我们选择了两个突变体，K698Q 和 K698N，作为进一步溶栓药物开发的候选药物。所选突变体可能是溶栓治疗的更好的治疗候选物。
