No abstract

High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.

Introduction: Anticoagulants, while an effective prophylactic for ischemic stroke increase a patient’s risk of major bleeding. There is currently an unmet need for new anticoagulants with the ideal combination of efficacy and low bleeding risk. Here we describe the in vitro and in vivo pharmacology of two development candidates VE-01902 and VE-02851, expected to enter phase I clinical trials in 2018 and 2019 respectively. These compounds belong to a new class of anti-coagulants, the PRecision Oral AntiCoagulants (PROACs), which inhibit fibrinogen cleavage at potencies comparable to the known Direct Thrombin Inhibitors (DTIs) while only weakly inhibiting thrombin induced platelet activation in plasma and whole blood. Methods: Enzyme activity assays by optical methods. Platelet status by expression of CD62P by flow cytometry. In vitro coagulation by Thrombin Generation Assay (TGA). In vivo efficacy and bleeding risk evaluated by rodent models of thrombosis and bleeding time tests. Results and Conclusions: The PROACs are a class of compounds that share a unique mechanism of action: reversible covalent inhibitors of thrombin with slow enzyme kinetics. We examined Thrombin Generation in platelet Poor Plasma (PPP) and Platelet Rich Plasma (PRP) of VE-01902 and VE-02851 and compared it to the known DTIs argatroban and dabigatran. In both plasma samples the PROACs potently inhibit the propagation phase (Endogenous Thrombin Potential (ETP)) while, unlike the known DTIs, only weakly affect the initiation phase of thrombin generation (10 to 20-fold weaker). In the PRP samples the PROACs do not significantly affect platelet activation even while suppressing ETP. To better understand the unique in vitro properties of the PROACs we studied the activity of multiple key factors of the coagulation cascade. Like argatroban and dabigatran, the PROACs protect mice from the effects of thrombin-induced pulmonary embolism and inhibit clot formation in the arteriovenous shunt rat model. However unlike these DTIs, they do not significantly inhibit platelet activation in either of these rodent models. Despite its strong anticoagulant properties, the PROACs show significantly lower bleeding than comparators.

We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.