Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Nail biting in rheumatic diseases.

To ascertain if nail biting (usually considered a manifestation of emotional tension) was associated with fibromyalgia, 387 patients attending the Rheumatism Clinic at the Leeds General Infirmary were studied prospectively. Bitten nails appeared to be a feature of youth rather than of fibromyalgia. Patients with a full set of dentures were less likely to bite their nails than others, but it can and was done by 7%. Posing the question about nail biting worried several patients, indicating the wisdom of seeking ethical approval even for "noninvasive" studies.

Thrombin inhibitors based on ketone derivatives of arginine and lysine.

Much attention is currently focused on inhibitors of thrombin as potential anticoagulants. We have previously reported thrombin inhibitors based on fragments of fibrinogen containing a ketomethylene isostere at P1-P'1. We now expand on these early findings by reporting on tripeptide based inhibitors of thrombin containing arginine or lysine ketones at the C-terminus. A large variety of such ketones have been studied and compared in their ability to increase the thrombin time in human plasma. In the case of arginine or lysine ketones the order of activity (i.e. decreasing IC50 TT) was: alkyl ketones < beta-ketoesters < difluoro beta-ketoamides < alkyloxymethyl ketones < fluoroketones. Lysine analogues were generally found to be ca. ten-fold less active than their arginine counterparts. However, in the case of alpha-ketoesters the lysine derivatives were superior to all the types of arginine ketones studied (including the arginine alpha-keto ester derived thrombin inhibitor). A mechanistic explanation of the relative inactivity of the arginine alpha-keto ester derivative is proposed. All the highly electrophilic ketones were found to be slow-binding with thrombin.

X-ray-crystallographic studies of complexes of pepstatin A and a statine-containing human renin inhibitor with endothiapepsin.

H-189, a synthetic human renin inhibitor, and pepstatin A, a naturally occurring inhibitor of aspartic proteinases, have been co-crystallized with the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6). H-189 [Pro-His-Pro-Phe-His-Sta-(statyl)-Val-Ile-His-Lys] is an analogue of human angiotensinogen. Pepstatin A [Iva(isovaleryl)-Val-Val-Sta-Ala-Sta] is a blocked pentapeptide which inhibits many aspartic proteinases. The structures of the complexes have been determined by X-ray diffraction and refined to crystallographic R-factors of 0.15 and 0.16 at resolutions of 0.18 nm (1.8 A) and 0.2 nm (2.0 A) respectively. H-189 is in an extended conformation, in which the statine residue is a dipeptide analogue of P1 and P'1 as indicated by the conformation and network of contacts and hydrogen bonds. Pepstatin A has an extended conformation to the P'2 alanine residue, but the leucyl side chain of the terminal statine residue binds back into the S'1 subsite, and an inverse gamma-turn occurs between P'1 and P'3. The hydroxy moiety of the statine at P1 in both complexes displaces the solvent molecule that hydrogen-bonds with the catalytic aspartate residues (32 and 215) in the native enzyme. Solvent molecules originally present in the native structure at the active site are displaced on inhibitor binding (12 when pepstatin A binds; 16 when H-189 binds).

A transition-state analogue inhibitor of human renin (H.261): test in vitro and a comparison with captopril in the anaesthetized baboon.

H.261, a new transition state inhibitor of human renin with an IC50 of 6.9 X 10(-10) M, was given by intravenous infusion to six anaesthetized baboons. The inhibitor was infused first at 0.1 mumol/kg/h for 15 min, then at 1.0 mumol/kg/h for a further 15 min. After a recovery period of 2 h in which the animals received 5% dextrose, they were infused with captopril, 25 mumol/kg/h for 15 min. At both rates of infusion H.261 markedly and significantly reduced the enzymatic action of renin in plasma, the blood concentration of angiotensin I, the plasma concentration of angiotensin II and mean arterial pressure. All changes reverted towards or to control values in the subsequent control period. Captopril also lowered plasma angiotensin II concentration and mean arterial pressure markedly and significantly but, as expected for an inhibitor of the angiotensin I-converting enzyme, plasma active renin concentration and blood angiotensin I concentration increased. The changes of angiotensin II and arterial pressure were similar with captopril and H.261.

Renin inhibitors: their use in understanding the role of angiotensin II as a pressor hormone.

Infusion of H.261, the inhibitor of human renin in the baboon, lowered blood angiotensin I, plasma angiotensin II, and arterial pressure suggesting that in the sodium-depleted state angiotensin II contributes to the maintenance of arterial pressure. In a second experiment dose-response infusions of angiotensin II were given in conscious sodium-depleted dogs before and during infusion of the renin inhibitor H.77. These suggested that the contribution of angiotensin II to the maintenance of arterial pressure in this state was made mainly by a circulating peptide. Preliminary results in normal humans show that infusion of H.142 intravenously lowered angiotensin I, angiotensin II, and arterial pressure.

New inhibitors of human renin tested in vitro and in vivo in the anaesthetized baboon.

A new inhibitor of human renin (H. 189) is described. It is a decapeptide analogue of human renin substrate with the amino acid, statine, substituted for leucine in the scissile bond. Its inhibitory potency as shown by IC50 is 1.0 X 10(-8) M with human plasma renin and 1.5 X 10(-8) M with baboon plasma renin. It is less effective with dog and rat renin, but its inhibitory potency with human renin is similar to that of another inhibitor of ours (H. 142) having a reduced isostere in the scissile bond. H. 189 has some inhibitory effect on cathepsin D (IC50 6.5 X 10(-5) M) but H. 142 has no discernible effect. Pepstatin, on the other hand, was highly effective against cathepsin D (IC50 1.2 X 10(-8) M). H. 142 and H. 189 were infused intravenously at 10 mg/kg/h in four anaesthetized salt-deplete baboons (Papio hamadryas). The activity of renin in plasma decreased markedly as did the circulating concentration of its products, angiotensin I and angiotensin II.