Derek Saunders

Covalent linking of photoreactive insulin to adipocytes produces a prolonged signal

The first step of insulins many cellular functions is specific binding to receptors on the plasma membrane of target cells. The subsequent molecular basis of insulin action, particularly the coupling mechanism(s) involved in transmitting the biological message, remains largely unknown1. Our approach to the problem centres on the application of a series of well characterized photo-insulins carrying an aryl–azido or nitro–aryl–azido group in positions A1, B1, B2 or B29 (refs 2–4). Specific binding to membrane components could be demonstrated with B1-, B29- (ref. 5) and A1-photo-insulins6 as well as a B2-derivative7. We now report that lipogenesis is increased to, and maintained at, near-maximal levels for several hours after photoinduced covalent binding of B2- (2-nitro,4-azidophenyl-acetyl)-des-PheB1-insulin (Napa-DP-insulin3) to living adipocytes.

Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Abstract Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [LeuB24]- and [LeuB25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature 28 :121–125). [LeuB24]- and [LeuB25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [LeuB24]-insulin to induce negative cooperativity was clearly impaired, that of [LeuB25]-insulin was almost abolished. [LeuB25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.

Discovery of Spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amines as Potent NOP and Opioid Receptor Agonists

We report the discovery of spiro[cyclohexane-pyrano[3,4-b]indole]-amines, as functional nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonists with strong efficacy in preclinical models of acute and neuropathic pain. Utilizing 4-(dimethylamino)-4-phenylcyclo-hexanone 1 and tryptophol in an oxa-Pictet-Spengler reaction led to the formation of spiroether 2, representing a novel NOP and opioid peptide receptor agonistic chemotype. This finding initially stems from the systematic derivatization of 1, which resulted in alcohols 3-5, ethers 6 and 7, amines 8-10, 22-24, and 26-28, amides 11 and 25, and urea 12, many with low nanomolar binding affinities at the NOP and mu opioid peptide (MOP) receptors.

Thrombin inhibitory and clot-specific fibrinolytic activities of the urokinase variant, M23 (rscu-PA-40 kDa/Hir)

The recombinant bifunctional urokinase variant, M23 (rscu-PA-40 kDA/Hir), comprising the kringle and protease domain of single-chain urokinase-type plasminogen activator and a C-terminal fragment of hirudin in one single-chain molecule, was evaluated for its thrombin-inhibitory and fibrinolytic properties in vitro and in vivo. M23 inhibited thrombin-activated coagulation of human blood and thrombin-induced aggregation of human platelet rich plasma in a concentration-dependent manner. The ADP-induced aggregation of human platelet rich plasma was not influenced by M23. In contrast, recombinant single-chain urokinase-type plasminogen activator (saruplase) inhibited neither blood coagulation nor platelet rich plasma aggregation. M23 and saruplase both lysed radiolabelled human thrombi immersed in human plasma (Chandler Loop system) with equal potency. However, there was a significantly lower systemic generation of plasmin (measured as consumption of alpha 2-antiplasmin) by M23 compared to saruplase. In anaesthetized non-heparinized rabbits, experimental femoral artery thrombosis was treated with intravenous bolus injections of M23 or saruplase (6 mg/kg, each). Thrombolytic restoration of arterial blood perfusion was significantly higher in M23- than in saruplase-treated rabbits. Plasma fibrinogen concentrations were decreased markedly in saruplase-treated animals, but remained at significantly higher levels in M23-treated rabbits. In conclusion, the bifunctional molecule, M23, showed thrombin inhibitory and fibrinolytic properties in human in vitro systems and exerted superior thrombolytic effects to saruplase in rabbit femoral artery thrombosis. In vitro and in vivo data indicate that the fibrinolytic activity of M23 is highly clot-specific.

Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development

Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.