Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface.

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 µM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 µM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain ß-strand to the core of the molecule. The release of the B-chain ß-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.

Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to l-homocysteine, yielding dimethylglycine and l-methionine. In this study, we prepared a new series of BHMT inhibitors. The inhibitors were designed to mimic the hypothetical transition state of BHMT substrates and consisted of analogues with NH, N(CH(3)), or N(CH(3))(2) groups separated from the homocysteine sulfur atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH(3)) moiety and ethylene spacer gave moderate inhibition. This result led us to prepare two inhibitors lacking a nitrogen atom in the S-linked alkyl chain: (RS,RS)-5-(3-amino-3-carboxypropylthio)-3-methylpentanoic acid and (RS)-5-(3-amino-3-carboxypropylthio)-3,3-dimethylpentanoic acid. Both of these compounds were highly potent inhibitors of BHMT. The finding that BHMT does not tolerate a true betaine mimic within these inhibitors, especially the nitrogen atom, is surprising and evokes questions about putative conformational changes of BHMT upon the binding of the substrates/products and inhibitors.

Insulin analogues with modifications at position B26. Divergence of binding affinity and biological activity.

In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and also with their ability to stimulate lipogenesis in vitro. We found that [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2 were very potent insulin analogues with respect to their binding affinities (214 and 465%, respectively, compared to that of human insulin), but they were significantly less potent than human insulin in vivo. Their full-length counterparts, [NMeHisB26]-insulin and [NMeAlaB26]-insulin, were less effective than human insulin with respect to binding affinity (10 and 21%, respectively) and in vivo activity, while [HisB26]-insulin exhibited properties similar to those of human insulin in all of the tests we carried out. The ability of selected analogues to stimulate lipogenesis in adipocytes was correlated with their biological potency in vivo. Taken together, our data suggest that the B26 residue and residues B26-B30 have ambiguous roles in binding affinity and in vivo activity. We hypothesize that our shortened analogues, [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2, have different modes of interaction with the insulin receptor compared with natural insulin and that these different modes of interaction result in a less effective metabolic response of the insulin receptor, despite the high binding potency of these analogues.

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.