Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Temporal relationship of sleep apnea and acromegaly: a nationwide study.

PURPOSE: Patients with acromegaly have an increased risk of sleep apnea, but reported prevalence rates vary largely. Here we aimed to evaluate the sleep apnea prevalence in a large national cohort of patients with acromegaly, to examine possible risk factors, and to assess the proportion of patients diagnosed with sleep apnea prior to acromegaly diagnosis. METHODS: Cross-sectional multicenter study of 259 Swedish patients with acromegaly. At patients' follow-up visits at the endocrine outpatient clinics of all seven university hospitals in Sweden, questionnaires were completed to assess previous sleep apnea diagnosis and treatment, cardiovascular diseases, smoking habits, anthropometric data, and S-IGF-1 levels. Daytime sleepiness was evaluated using the Epworth Sleepiness Scale. Patients suspected to have undiagnosed sleep apnea were referred for sleep apnea investigations. RESULTS: Of the 259 participants, 75 (29%) were diagnosed with sleep apnea before the study start. In 43 (57%) of these patients, sleep apnea had been diagnosed before the diagnosis of acromegaly. After clinical assessment and sleep studies, sleep apnea was diagnosed in an additional 20 patients, yielding a total sleep apnea prevalence of 37%. Higher sleep apnea risk was associated with higher BMI, waist circumference, and index finger circumference. Sleep apnea was more frequent among patients with S-IGF-1 levels in the highest quartile. CONCLUSION: Sleep apnea is common among patients with acromegaly, and is often diagnosed prior to their acromegaly diagnosis. These results support early screening for sleep apnea in patients with acromegaly and awareness for acromegaly in patients with sleep apnea.

Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice.

The efficacy of islet transplantation for diabetes treatment suffers from lack of cadaver-derived islets, islet necrosis and long transfer times prior to transplantation. Here, we developed a method for culturing mouse and human islets in vitro on α5-laminins, which are natural components of islet basement membranes. Adhering islets spread to form layers of 1-3 cells in thickness and remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 h. Transplantation of 110-150 mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3-7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes. RNA-sequencing of isolated and cultured mouse islets provided further evidence for the adhesion and spreading achieved with α5-laminin. Our results suggest that use of such in vitro expanded islets may significantly enhance the efficacy of islet transplantation treatment for diabetes.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Association of genetic variants at 3q22 with nephropathy in patients with type 1 diabetes mellitus.

Diabetic nephropathy (DN) is the primary cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM) and affects about 30% of these patients. We have previously localized a DN locus on chromosome 3q with suggestive linkage in Finnish individuals. Linkage to this region has also been reported earlier by several other groups. To fine map this locus, we conducted a multistage case-control association study in T1DM patients, comprising 1822 cases with nephropathy and 1874 T1DM patients free of nephropathy, from Finland, Iceland, and the British Isles. At the screening stage, we genotyped 3072 tag SNPs, spanning a 28 Mb region, in 234 patients and 215 controls from Finland. SNPs that met the significance threshold of p < 0.01 at this stage were followed up by a series of sample sets. A genetic variant, rs1866813, in the noncoding region at 3q22 was associated with increased risk of DN (overall p = 7.07 x 10(-6), combined odds ratio [OR] of the allele = 1.33). The estimated genotypic ORs of this variant in all Finnish samples suggested a codominant effect, resulting in significant association, with a p value of 4.7 x 10(-5) (OR = 1.38; 95% confidence interval = 1.18-1.62). Additionally, an 11 kb segment flanked by rs62408925 and rs1866813, two strongly correlated variants (r(2) = 0.95), contains three elements highly conserved across multiple species. Independent replication will clarify the role of the associated variants at 3q22 in influencing the risk of DN.

Evaluation of ((4-hydroxyphenyl)ethyl)maleimide for site-specific radiobromination of anti-HER2 affibody.

Affibody molecules are a new class of small phage-display selected proteins using a scaffold domain of the bacterial receptor protein A. They can be selected for specific binding to a large variety of protein targets. An affibody molecule binding with high affinity to a tumor antigen HER2 was recently developed for radionuclide diagnostics and therapy in vivo. The use of the positron-emitting nuclide (76)Br (T(1/2) = 16.2 h) could improve the sensitivity of detection of HER2-expressing tumors. A site-specific radiobromination of a cysteine-containing variant of the anti-HER2 affibody, (Z(HER2:4))(2)-Cys, using ((4-hydroxyphenyl)ethyl)maleimide (HPEM), was evaluated in this study. It was found that HPEM can be radiobrominated with an efficiency of 83 +/- 0.4% and thereafter coupled to freshly reduced affibody with a yield of 65.3 +/- 3.9%. A "one-pot" labeling enabled the radiochemical purity of the conjugate to exceed 97%. The label was stable against challenge with large excess of nonlabeled bromide and in a high molar strength solution. In vitro cell tests demonstrated that radiobrominated affibody binds specifically to the HER2-expressing cell-line, SK-OV-3. Biodistribution studies in nude mice bearing SK-OV-3 xenografts have shown tumor accumulation of 4.8 +/- 2.2% IA/g and good tumor-to-normal tissue ratios.

Survivors of childhood acute lymphoblastic leukaemia, with radiation-induced GH deficiency, exhibit hyperleptinaemia and impaired insulin sensitivity, unaffected by 12 months of GH treatment.

OBJECTIVE: Adult survivors of childhood acute lymphoblastic leukaemia (ALL) often exhibit GH deficiency (GHD), due to prophylactic cranial radiotherapy (CRT). It is not known whether the observed risk for adiposity in these patients is associated with impaired insulin sensitivity and whether the insulin sensitivity is affected by GH replacement therapy. SUBJECTS AND DESIGN: Eleven patients with GHD (median age 29 years), previously given prophylactic CRT for ALL, and 11 sex-, age- and body mass index (BMI)-matched controls were investigated with bioimpedance analysis (BIA) and analysis of serum leptin, serum free fatty acids (FFA) and serum insulin. Insulin sensitivity was measured by a euglycaemic-hyperinsulinaemic clamp technique (IS-clamp). Moreover, the effects of 12 months of individually titrated GH treatment (median dose 0.5 mg/day) on these parameters were investigated. RESULTS: At baseline, the patients had lower fat free mass (FFM) (P = 0.003), higher percentage fat mass (FM) (P = 0.05), serum insulin (P = 0.02) and serum leptin/kg FM (P = 0.01) than controls. The patients had a tendency towards impaired IS-clamp (P = 0.06), which disappeared after correction for body composition (IS-clamp/kg FFM; P > 0.5). In the patients, time since CRT was positively correlated with percentage FM (r = 0.70, P = 0.02), and there was an independent negative association between serum FFA and IS-clamp (P = 0.05). Twelve months of GH treatment increased serum IGF-I (P = 0.003) and FFM (P = 0.02) and decreased percentage FM (P = 0.03), but no significant changes were seen in serum leptin/kg FM, serum FFA, FFA-clamp, serum insulin or IS-clamp (all, P > or = 0.2). CONCLUSIONS: Young adult survivors of childhood ALL with GHD had increased fat mass, hyperleptinaemia and impaired insulin sensitivity, which could be a consequence of radiation-induced impairment of GH secretion or mediated by other hypothalamic dysfunctions, such as leptin resistance or other unknown factors, affected by CRT. Twelve months of individualized GH replacement therapy led to positive effects on body composition, but the hyperleptinaemia, hyperinsulinaemia and the impaired insulin sensitivity remained unchanged.