The semisynthesis of octadeutero-PheB1-octadeutero-ValB2]-procine insulin and its characterization by mass spectrometry

Insulin analogues labelled with stable isotopes (e.g. deuterium, 18O, 15N, etc.) are authentic (the native structure is rigorously maintained), non-radioactive (preferred for injection into man) and can easily be distinguished from endogenous insulin by mass spectrometry by virtue of their molecular masses. Appropriate combinations of amino-protecting groups (methylsulphonylethyloxycarbonyl and t-butoxy carbonyl), Edman degradation and chemical coupling were used to produce [octadeutero-PheB1]-porcine insulin and [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin. The analogues were characterized by electrospray ionization mass spectrometry. Standard mixtures of labelled and unlabelled insulins were successfully studied by mass spectrometry. Isotope dilution mass spectrometry could therefore provide a useful direct measure of insulin under true physiological conditions, without many of the drawbacks of existing methods. In this regard, the analogue with 16 deuteriums was more suitable than the octadeuterated analogue, since the greater mass difference between the labelled and unlabelled forms enabled a lower mass spectrometric resolution to be used, resulting in higher sensitivit

Enzymatic semisynthesis of insulin specifically labelled with tritium at position B-30

We have synthesized porcine insulin labelled with tritium at position B-30 using enzyme-catalysed formation of a peptide bond. The resulting insulin derivative has the label in the expected position and is biologically active. We have tested our procedure to prepare batches up to 50μCi of tritiated insulin at a specific radioactivity of up to 1.14 Ci/mmo

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry

The production of semisynthetic human insulin for therapeutic purposes is of considerable importance. During trypsin-catalysed transformation of pig insulin into an ester of insulin of human sequence, the alanyl residue at position B30 is removed and replaced with an esterified residue of threonine. We have carried out this transformation in a medium enriched in 18OH2 and studied the product by MS. In contrast to a previous report, we find that incorporation of label into the B29−B30 peptide bond occurs during the transformation with threonine methyl ester in aqueous N, N-dimethylacetamide. Quantitative data are presented and the implications of these findings are discusse

Highly potent HIV inhibition: engineering a key anti-HIV structure from PSC-RANTES into MIP-1β/CCL4

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1β/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1β/CCL4 analogues that retain the receptor binding profile of MIP-1β/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potenc

Topically Applied Recombinant Chemokine Analogues Fully Protect Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge

Effective strategies for preventing human immunodeficiency virus infection are urgently needed, but recent failures in key clinical trials of vaccines and microbicides highlight the need for new approaches validated in relevant animal models. Here, we show that 2 new chemokine (C-C motif) receptor 5 inhibitors, 5P12-RANTES (regulated on activation, normal T cell expressed and secreted) and 6P4-RANTES, fully protect against infection in the rhesus vaginal challenge model. These highly potent molecules, which are amenable to low-cost production, represent promising new additions to the microbicides pipelin

Resistance to the CCR5 Inhibitor 5P12-RANTES Requires a Difficult Evolution from CCR5 to CXCR4 Coreceptor Use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16–18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a “resistant” variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching