Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors

Bovine seminal ribonuclease (BS-RNase) is a 27 kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2′-phosphate (U2′p), uridine 3′-phosphate (U3′p), uridine 5′-diphosphate (UDP), cytidine 3′-phosphate (C3′p), and cytidine 5-phosphate (C5′p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3′p being the most potent with a Ki value of 22 μM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2′p, U3′p, and C5′p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3′p and C5′p inhibit significantly this biological action of BS-RNase

Targeted Polymer-Based Probes for Fluorescence Guided Visualization and Potential Surgery of EGFR-Positive Head-and-Neck Tumors

This report describes the design, synthesis and evaluation of tumor-targeted polymer probes to visualize epidermal growth factor receptor (EGFR)-positive malignant tumors for successful resection via fluorescence guided endoscopic surgery. Fluorescent polymer probes of various molecular weights enabling passive accumulation in tumors via enhanced permeability and retention were prepared and evaluated, showing an optimal molecular weight of 200,000 g/mol for passive tumor targeting. Moreover, poly(N-(2-hydroxypropyl)methacrylamide)-based copolymers labeled with fluorescent dyes were targeted with the EGFR-binding oligopeptide GE-11 (YHWYGYTPQNVI), human EGF or anti-EGFR monoclonal antibody cetuximab were all able to actively target the surface of EGFR-positive tumor cells. Nanoprobes targeted with GE-11 and cetuximab showed the best targeting profile but differed in their tumor accumulation kinetics. Cetuximab increased tumor accumulation after 15 min, whereas GE 11 needed at least 4 h. Interestingly, after 4 h, there were no significant differences in tumor targeting, indicating the potential of oligopeptide targeting for fluorescence-navigated surgery. In conclusion, fluorescent polymer probes targeted by oligopeptide GE-11 or whole antibody are excellent tools for surgical navigation during oncological surgery of head and neck squamous cell carcinoma, due to their relatively simple design, synthesis and cost, as well as optimal pharmacokinetics and accumulation in tumors