Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.

Free submandibular gland autotransplantation is used to treat absolute tear deficiency. Although disconnected from any peripheral innervation, most transplants show increasing secretion for years. We have evaluated the secretory activity and autonomic innervation of such transplants. Secretory activity of glands in response to parasympatholytics and parasympathomimetics was evaluated by Schirmer's test and Technetium scintigraphy. Submandibular gland tissue specimens taken before and after transplantation were examined histologically. Relative hypersecretion during the first postoperative week suddenly decreased but then slowly increased during the first postoperative year. Hypersecretion was significantly reduced by parasympatholytics while carbachol rapidly increased secretion. Histology of transplanted glandular tissue showed parenchymal atrophy. Cholinesterase-positive nerves were abundant and in a similar distribution to normal with scattered positive ganglion cells. Adrenergic axons were fewer than normal and irregularly distributed. Early hypersecretion may be due to release of neurotransmitters from degenerating terminal axons. This is followed by a period of minimal secretion during which hypersensitivity of acinar cells develops. With spontaneous reinnervation, secretion is accentuated by external sympathetic vasomotor adrenergic drive. This shows that submandibular glands can remain viable despite complete separation from their normal nerve supply and are capable of regaining a substantial secretory activity for years.

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SUMMARYChanges in serum levels of rat tissue kalljkrein (rKl) in venous blood were measured, using a newly developed radioimmunoassay, before and after autonomic nerve stimulations of submandibular salivary secretion. rK1 secreted into saliva under these conditions was measured by radioimmunoassay and by enzymic activity assay, using the fluorogenic peptide substrate D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC). Following an overnight fast, serum rK I concentration was 30-40 ng ml-'. Unilateral electrical stimulation of the submandibular sympathetic nerve supply (at 50 Hz in bursts of 1 s every 10 s for 60 min) evoked a small flow of saliva with a very high rK I concentration, resulting in a large output of rK l of 2104.4 + 603 5 jug (n = 6). Such stimulation caused a large degranulation of granular duct cells and a corresponding reduction in glandular rK 1 content. Unilateral electrical stimulation of the parasympathetic nerve supply (at 5 Hz continuously for 60 min) evoked a copious flow of saliva with a very low rK1 concentration, resulting in a low output of rK 1 (18 1 + 4.9 jug; n = 6). Despite these large differences in salivary outputs of rK1, serum concentrations of rK1 were increased similarly following either sympathetic or parasympathetic stimulation by 48 and 46 %, respectively. If the submandibular duct was briefly obstructed during sympathetic stimulation, inducing leakage and glandular oedema, then serum rK I increased greatly (40-fold); a similar increase to that seen by others in previous studies without deliberate obstruction. Four days after bilateral submandibular-sublingual sialadenectomy serum rK 1 concentration was reduced by approximately 50 %. The results indicate that submandibular glands normally contribute to circulating levels of rKl in rats, but this contribution is independent of the amounts of rKl secreted into saliva by sympathetically induced exocytosis, and is likely to arise from basal vesicular transport. However, if glandular leakage occurs during sympathetic stimulation of submandibular secretion this then causes increases in the circulating levels of rK1 that correlate with the large amounts being secreted into saliva.