The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephroand ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of '"I-urokinase-plasminogen activator inhibitor type-i complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 ,uM for aprotinin, 50 tiM for gentamicin, and 0.5 tiM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H]gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human a2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target.In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity. (J. Clin.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha‐MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post‐translational processing of these mutants and have also studied two C‐terminal v‐rasH point mutants: one encodes serine in place of cysteine‐186, the other threonine for valine‐187. The Thr‐187 mutant was transformation‐competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation‐competent deletion mutant from which amino acids 166‐175 had been deleted. The Ser‐186 mutant was defective for transformation. The p21s encoded by the Ser‐186 mutant and by the previously described transformation‐defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell‐encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp 181 , Lys 120 , and Tyr 46 . PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.