The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine, a cell-penetrating peptide

Some Vinca alkaloids (e.g. vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra-, hexa- or octapeptides) cell-penetrating peptides on the cytostatic activity in vitro and in vivo. Br-Vindoline-(L)-Trp-OH attached to the N-terminus of octaarginine was the most effective compound in vitro on HL-60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br-vindoline-(L)-Trp-Arg8 and Br-vindoline-(D)-Trp-Arg8 suggest the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced of the activity on C26 and P388 cells: conjugate with (L)-Trp was more active than conjugate with the (D)-isomer. In contrast conjugates had very similar effect on both the HL-60 and MDA-MB-231 cells. In preliminary experiments conjugate Br-vindoline-(L)-Trp-Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor-bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity

Optimal collision energies and bioinformatics tools for efficient bottom-up sequence validation of monoclonal antibodies

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biophar-maceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of MS/MS collision energies and automated combina-tion of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called “Serac”) that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of se-quence coverage (we term this “confirmed sequence coverage”). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2–3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields about 85% confirmed sequence coverage, 25–30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative di-gestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences