The discovery of novel, potent and highly selective inhibitors of inducible nitric oxide synthase (iNOS).

By careful analysis of experimental X-ray ligand crystallographic protein data across several inhibitor series we have discovered a novel, potent and selective series of iNOS inhibitors exemplified by compound 8.

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase.

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.

Beliefs about health and illness in women managed for gestational diabetes in two organisations.

OBJECTIVE: to explore beliefs about health, illness and health care in women with gestational diabetes mellitus (GDM) managed in two different organisations based on diabetology or obstetrics. DESIGN: an explorative qualitative study using semi-structured interviews. SETTING: clinic A: a specialist diabetes clinic with regular contact with a diabetologist and antenatal care provided by a midwife; clinic B: a specialist maternity clinic providing regular contact with a midwife, a structured programme for self-monitoring of blood glucose and insulin treatment, and a 1-day diabetes class by an obstetrician, a diabetologist, a midwife and a dietician. The clinics were located at two different university hospitals in Sweden. PARTICIPANTS: a consecutive sample of Swedish women diagnosed with GDM; 13 managed in clinic A and 10 managed in clinic B. MEASUREMENT AND FINDINGS: women described their perceptions of as well-being, being healthy and freedom from disease. All respondents reported a delay in the provision of information about GMD and an information gap about GDM and the management of the condition, from diagnosis until the start of treatment at the specialist clinic. Respondents from clinic A expressed fear about future development of type 2 diabetes. Women from clinic B discussed different causes of GDM, and many claimed that health-care staff informed them that GDM was a transient condition during pregnancy. Respondents from clinic A reported a conflict in their treatment of pregnancy and GDM as two different conditions. KEY CONCLUSIONS: beliefs differed and were related to the health-care model chosen. Women with GDM monitored at a specialist maternity clinic believed GDM to be a transient condition during pregnancy only, whereas women monitored at a diabetes specialist clinic expressed fear about a future risk of developing type 2 diabetes. IMPLICATIONS FOR PRACTICE: relevant information about GDM should be provided without delay after initial diagnosis and thereafter repeatedly. It is important to recognise the context of information given on GDM, as it will substantially influence the beliefs and attitudes of women towards GDM as a transient condition during pregnancy or as a potential risk factor for diabetes.

A simplified oral glucose tolerance test in pregnancy: compliance and results.

BACKGROUND: To describe a reliable method for a general oral glucose tolerance test (OGTT) during pregnancy, to evaluate adherence to the method, and to compare the frequency of reported gestational diabetes mellitus (GDM) and perinatal outcome in affected pregnancies in Skåne, using direct diagnostic OGTT, with those from a comparable area, Halmstad-Ljungby-Växjö (HLV), using random glucose measurements (RGM) to identify women for the OGTT. METHODS: The OGTT program and quality assurance in Skåne is described. Antenatal records on deliveries in May 2003 were scrutinised to ascertain if OGTT had been performed. Frequencies of GDM, prematurity and large for gestational age (LGA) infants were estimated using a population-based perinatal database (PRS). RESULTS: OGTT was performed in 93% of pregnant women in Skåne. In 2000-2003 GDM frequency in Skåne was twice as high as in HLV (1.9 versus 1%), while the frequency of LGA and prematurity among infants of mothers who were diagnosed with GDM were similar. CONCLUSIONS: Decentralised general OGTT is a reliable and effective method to diagnose GDM. OGTT is twice as sensitive as RGM, and the severity of GDM in the cases identified with OGTT did not differ from the severity of those identified with RGM.

Screening for MODY mutations, GAD antibodies, and type 1 diabetes--associated HLA genotypes in women with gestational diabetes mellitus.

OBJECTIVE: To investigate whether genetic susceptibility to type 1 diabetes or maturity-onset diabetes of the young (MODY) increases susceptibility to gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS: We studied mutations in MODY1-4 genes, the presence of GAD antibodies, and HLA DQB1 risk genotypes in 66 Swedish women with GDM and a family history of diabetes. An oral glucose tolerance test was repeated in 46 women at 1 year postpartum. RESULTS: There was no increase in type 1 diabetes-associated HLA-DQB1 alleles or GAD antibodies when compared with a group of type 2 diabetic patients (n = 82) or healthy control subjects (n = 86). Mutations in known MODY genes were identified in 3 of the 66 subjects (1 MODY2, 1 MODY3, and 1 MODY4). Of the 46 GDM subjects, 2 had diabetes (4%) and 17 had impaired glucose tolerance (IGT) (37%) at 1 year postpartum. Of the two subjects who developed manifest diabetes, one carried a MODY3 mutation (A203H in the hepatocyte nuclear factor-1alpha gene). There was no increase in high-risk HLA alleles or GAD antibodies in the women who had manifest diabetes or IGT at 1 year postpartum. CONCLUSIONS: MODY mutations but not autoimmunity contribute to GDM in Swedish women with a family history of diabetes and increase the risk of subsequent diabetes.

2-aminopyridines as highly selective inducible nitric oxide synthase inhibitors. Differential binding modes dependent on nitrogen substitution.

4-Methylaminopyridine (4-MAP) (5) is a potent but nonselective nitric oxide synthase (NOS) inhibitor. While simple N-methylation in this series results in poor activity, more elaborate N-substitution such as with 4-piperidine carbamate or amide results in potent and selective inducible NOS inhibition. Evidently, a flipping of the pyridine ring between these new inhibitors allows the piperidine to interact with different residues and confer excellent selectivity.

Different HLA-DR-DQ and MHC class I chain-related gene A (MICA) genotypes in autoimmune and nonautoimmune gestational diabetes in a Swedish population.

The genetic susceptibility for gestational diabetes (GDM) was estimated by comparisons of genotypes within human leukocyte antigen (HLA) and major histocompatibility complex class I chain-related gene A (MICA) in 199 women with GDM and 213 healthy women. At least one of ICA, glutamic acid decarboxylase antibodies, or islet cell antigen-2 antibodies/tyrosine phosphatase antibodies was found in 6.0% (12/199) of women with GDM and were considered as autoimmune GDM, whereas the remaining 187 were considered as nonautoimmune GDM. HLA genotyping was done with polymerase chain reaction and sequence-specific oligonucleotides. MICA polymorphism was determined with polymerase chain reaction and fragment size determination. HLA-DR3-DQ2/x or DR4-DQ8/x and MICA5.0/5.1 were more frequent in autoimmune GDM compared with controls; 92% versus 46% and 42% versus 13% and conferred increased risk (odds ratio [OR] = 13; 95% confidence interval [CI] 1.7-104) and (OR = 4.7; 95%CI 1.4-16). Four other genotypes were more frequent in nonautoimmune GDM compared with controls: HLA-DR7-DQ2/y, 24% versus 14%; DR9-DQ9/y, 9.6% versus 1.9%; DR14-DQ5/y, 7.5% versus 0.94%; and MICA5.0/z, 24% versus 13% and gave increased risk: OR = 2.0; 95%CI 1.2-3.4, OR = 5.6; 95%CI 1.8-17, OR = 8.5; 95%CI 1.9-38, and OR = 2.0; 95%CI 1.2-3.4, respectively. We concluded that autoimmune diabetes with onset during pregnancy is associated with the type 1 diabetes-associated genotypes and also with MICA5.0/5.1, whereas DR7-DQ2/y, DR9-DQ9/y, DR14-DQ5/y, and MICA5.0/z are risk factors for nonautoimmune GDM.

Changes in lipid and lipoprotein profile in postmenopausal women receiving low-dose combinations of 17beta-estradiol and norethisterone acetate.

OBJECTIVE: To evaluate the modification of lipid and lipoprotein by use of low doses of continuous-combined formulations of 17beta-estradiol (E ) and norethisterone acetate (NETA) in healthy postmenopausal women. DESIGN: The study was designed as a double-blind, randomized, placebo-controlled trial. A total of 120 healthy postmenopausal women were randomized to one of three treatment arms: (1) placebo group ( = 40); (2) E /NETA 0.25-mg group-subjects receiving oral continuous-combined E 1 mg and NETA 0.25 mg ( = 40); (3) E /NETA 0.5-mg group-women who were treated with E 1 mg and NETA 0.5 mg ( = 40). The duration of study was 12 months. Plasma levels of total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL) cholesterol, triglycerides, lipoprotein(a), apolipoprotein A and apolipoprotein B were determined on four occasions (i.e., baseline, 3-, 6-, and 12-month visits). RESULTS: There were no differences in the baseline characteristics among the three groups. A total of 102 women completed the study, resulting in a compliance rate of 85%. There was a significant reduction of total cholesterol, LDL cholesterol, and lipoprotein(a) in both combined groups when compared with placebo. The level of apolipoprotein B declined significantly only in the E /NETA 0.25-mg group. Decrements were observed within 3 months of treatment and maintained thereafter. No significant changes were found in triglycerides, VLDL cholesterol, HDL cholesterol, apolipoprotein A, and LDL/HDL ratio. Between the two active combined groups, no statistically significant differences were noted. CONCLUSION: Favorable changes in lipids and lipoproteins were associated with the low dose of E /NETA combinations. These effects may contribute to the reduction or prevention of atherogenesis in postmenopausal women.

Low-dose hormone therapy and carbohydrate metabolism.

OBJECTIVE: To evaluate the influence by two low doses of oral continuous-combined formulations of 17 beta-estradiol (E(2)) and norethisterone acetate (NETA) on carbohydrate metabolism in healthy postmenopausal women. DESIGN: A double-blind, randomized, placebo-controlled trial. SETTING: Volunteers at a university hospital. SUBJECT: One hundred twenty healthy postmenopausal women. INTERVENTION(S): One hundred twenty women were randomized to three treatment arms: (1) E(2) 1 mg/NETA 0.25 mg group (n = 40); (2) E(2) 1 mg/NETA 0.5 mg group (n = 40); (3) placebo group (n = 40). A total of 102 women completed 12 months of treatment. An oral glucose tolerance test (OGTT) was performed at baseline and at 3, 6, and 12 months. MAIN OUTCOME MEASURE(S): Fasting glucose, fasting insulin, total area under the curve (AUC) and insulin/glucose index during OGTT. RESULT(S): Fasting levels of glucose and insulin declined significantly in the E(2)/NETA 0.5 mg group. At OGTT, the total AUC for insulin declined in both active arms. The curve for glucose increased significantly in the E(2)/NETA 0.25 mg group. A lower insulin/glucose index was observed during OGTT in both active regimens when compared with placebo. In the active treatment groups, a significant reduction of fasting glucose and/or fasting insulin was encountered in women with higher basal fasting levels (fasting glucose >4.2 mmol/L or log-fasting insulin >0.87). CONCLUSION(S): Oral low-dose E(2) 1 mg/NETA 0.5 mg regimen did not impair carbohydrate metabolism, but seemed to improve insulin sensitivity in healthy postmenopausal women.

Efficiency of hit generation and structural characterization in fragment-based ligand discovery.

Fragment-based ligand discovery constitutes a useful strategy for the generation of high affinity ligands with suitable physico-chemical properties to serve as drug leads. There is an increasing number of generic biophysical screening strategies established with the potential for accelerating the generation of useful fragment hits. Crystal structures of these hits can subsequently be used as starting points for fragment evolution to high affinity ligands. Emerging understanding of the efficiency and operative aspects of hit generation and structural characterization in FBLD suggests that this method should be well suited for academic ligand development of chemical tools and experimental therapeutics.

Abnormal fetal growth is associated with gestational diabetes mellitus later in life: population-based register study.

BACKGROUND: Increasing evidence has been collected that intrauterine growth restriction is associated with development of type-2 diabetes mellitus in adult life. The present study was designed to test the hypothesis that abnormal intrauterine growth of female fetuses correlates with their future risk of developing gestational diabetes mellitus (GDM). METHODS: Population-based register study of the data from the Swedish Medical Birth Registry; perinatal data from 1973 to 1983 were linked with the diagnosis of GDM during 1987-2001. 421 women with GDM diagnosis were compared to 60,890 controls with regard to maternal age and parity, maternal education (data from the Education Register of Statistics Sweden), maternal diagnosis of diabetes, gestational duration, birth weight, and gestational age-related birth weight. RESULTS: There was a significant association between low (odds ratio (OR): 2.15, 95% confidence interval (CI): 1.29-3.50), as well as high (OR: 1.97, 95% CI: 1.12-3.45) birth weight and later development of GDM. There was a U-shaped relation between the gestational age-related birth weight and risk of developing GDM. A young mother and prematurity increased the risk of the offspring developing GDM. Nine of the 421 women (2.1%) with GDM were born to mothers who had a diagnosis of diabetes. The corresponding figures for the controls were 205 of 60,890 (0.3%). CONCLUSION: Intrauterine conditions and/or genetic disposition, which affect prenatal growth, increase the future risk of the female fetus developing GDM.

Presence of GAD antibodies during gestational diabetes mellitus predicts type 1 diabetes.

OBJECTIVE: We sought to study the frequency of beta-cell-specific autoantibody markers in women with gestational diabetes mellitus (GDM) and to follow these women to estimate the risk of later development of type 1 diabetes. RESEARCH DESIGN AND METHODS: Of 385 pregnant women with GDM during 1995-2005 in the district of Lund, 24 (6%) women were found positive for at least one of the following: islet cell antibody (ICA), GAD antibody (GADA), or tyrosine phosphatase antibody (IA-2A). The women were followed and autoantibodies reanalyzed. Those who had not developed diabetes did an oral glucose tolerance test. The frequencies of known risk factors for GDM were compared in women with GDM with and without pancreatic autoantibodies. RESULTS: Among the autoantibody-positive women, 50% had developed type 1 diabetes compared with none among the GDM control subjects (P = 0.001), 21% had impaired fasting glucose or impaired glucose tolerance compared with 12.5% among control subjects (P = 0.3), and none had developed type 2 diabetes compared with 12.5% among control subjects (P = 0.1). CONCLUSIONS: Autoantibody screening in pregnant women with GDM and follow-up after delivery should be considered for early recognition of type 1 diabetes.

Pregnancy outcome after maternal organ transplantation in Sweden.

OBJECTIVE: To study pregnancy outcome before and after organ transplantation. DESIGN: Registry study. SETTING: Swedish Health Registers. POPULATION: All births in Sweden 1973-2002. METHODS: Women who had organ transplantation were identified from the Hospital Discharge Register and their deliveries before and after the transplantation were identified from the Medical Birth Register. Abortions requiring hospitalisation were identified from the Hospital Discharge Register. Outcomes were compared with those in the total population. Adjustments were made for maternal age, parity and smoking habits. MAIN OUTCOME MEASURES: Miscarriage, preterm delivery, low birthweight, small for gestational age, congenital malformations, infant death, pre-eclampsia and placental abruption. RESULTS: A total of 980 infants born before and 152 born after transplantation were identified. A high frequency of pre-eclampsia (22%), preterm birth (46%), low birthweight (41%), small for gestational age (16%) and infant death (5% before the age of one) were found for deliveries after transplantation but similar frequencies were found also among deliveries a few years before transplantation. No significant increase in congenital malformation rate was seen. Among 15 infants born after liver transplantation, 2 were malformed. CONCLUSIONS: Pregnancies after organ transplantation have an increased risk of complications but this is similar to pregnancies occurring before the transplantation.

Relationship between vitamin use, smoking, and nausea and vomiting of pregnancy.

BACKGROUND: Nausea and vomiting in pregnancy (NVP) is a common complaint but risk factors for NVP are not well characterized. METHODS: Occurrence of NVP was studied by questionnaires given to pregnant women at their first visit to the antenatal care unit and were returned around gestational week 28. RESULTS: Analysis of 3675 completed questionnaires was made. Nausea and vomiting in pregnancy was reported by 79% of the women, approximately half of which had been vomiting. Various therapies (drugs, acupuncture, acupressure) were tried by 18% of the women with NVP, of which the majority used drugs, most notably antihistamines (specifically meclozine). Hospitalization occurred in 1% of all women. Nausea and vomiting in pregnancy caused 28% of all sick-leaves during the first 28 weeks of pregnancy. Low maternal age and parity 1+ independently increased the risk for NVP. Smoking before pregnancy and using vitamins in early pregnancy were associated with a decreased risk for NVP. Women working outside the home had a lower rate of NVP than housewives and women out of work. CONCLUSIONS: Nausea and vomiting in pregnancy is a common complaint with a significant impact on leave of absence from work. The study identifies a number of factors that are related to the occurrence of NVP and that may give hints on the etiology of the condition.

Acupuncture relieves pelvic and low-back pain in late pregnancy.

BACKGROUND: The study was designed to evaluate the analgesic effect and possible adverse effects of acupuncture for pelvic and low-back pain during the last trimester of pregnancy. METHODS: Following individual informed consent, 72 pregnant women reporting pelvic or low-back pain were randomized during pregnancy weeks 24-37 to an acupuncture group (n = 37) or to a control group (n = 35) at three maternity wards in southern Sweden. Traditional acupuncture points and local tender points (TP) were chosen according to individual pain patterns and stimulated once or twice a week until delivery or complete recovery in acupuncture patients. Control patients were given no sham stimulation. Throughout the study period each patient made weekly visual analog scale (VAS) evaluations of maximal and minimal pain intensity as well as three-point assessments of pain intensity during various activities. RESULTS: During the study period, VAS scorings of pain intensity decreased over time in 60% of patients in the acupuncture group and in 14% of those in the control group (p < 0.01). At the end of the study period, 43% of the acupuncture patients were less bothered than initially by pain during activity compared with 9% of control patients (p < 0.01). No serious adverse effects of acupuncture were found in the patients, and there were no adverse effects at all in the infants. CONCLUSION: Acupuncture relieves low-back and pelvic pain without serious adverse effects in late pregnancy.

Predictive factors of developing diabetes mellitus in women with gestational diabetes.

BACKGROUND: To investigate which factors during gestational diabetes pregnancies correlate with the risk of developing impaired glucose tolerance or diabetes 1 year postpartum and to compare this risk in women with gestational diabetes and women with a normal oral glucose tolerance test during pregnancy. METHODS: Of 315 women with gestational diabetes, defined as a 2-hr blood glucose value of at least 9.0 mmol/l at a 75-g oral glucose tolerance test, who delivered in Lund 1991-99, 229 (73%) performed a new test 1 year postpartum. We compared maternal and fetal factors during pregnancy with the test value at follow up. A control group of 153 women with a 2-hr test value below 7.8 mmol/l during pregnancy were invited to a new test 1 year postpartum and 60 (39%) accepted. RESULTS: At 1 year follow up, 31% of the women with gestational diabetes but only one of the 60 controls showed pathologic glucose tolerance and one had developed diabetes. The following factors in women with gestational diabetes were identified as predicting impaired glucose tolerance or diabetes at 1 year follow up: maternal age over 40 and--in a multiple regression analysis, independent of each other--a high 2-hr value at oral glucose tolerance test during pregnancy and insulin treatment during pregnancy. CONCLUSION: The risk of developing manifest diabetes after gestational diabetes may be high enough to justify a general screening or diagnostic procedure in all pregnant women to identify women with gestational diabetes and a postpartum follow up program for them. This study did not identify any particular factor during pregnancy with enough precision to predict a later progression to diabetes.

Conformational changes in nitric oxide synthases induced by chlorzoxazone and nitroindazoles: crystallographic and computational analyses of inhibitor potency.

Nitric oxide is a key signaling molecule in many biological processes, making regulation of nitric oxide levels highly desirable for human medicine and for advancing our understanding of basic physiology. Designing inhibitors to specifically target one of the three nitric oxide synthase (NOS) isozymes that form nitric oxide from the L-Arg substrate poses a significant challenge due to the overwhelmingly conserved active sites. We report here 10 new X-ray crystallographic structures of inducible and endothelial NOS oxygenase domains cocrystallized with chlorzoxazone and four nitroindazoles: 5-nitroindazole, 6-nitroindazole, 7-nitroindazole, and 3-bromo-7-nitroindazole. Each of these bicyclic aromatic inhibitors has only one hydrogen bond donor and therefore cannot form the bidentate hydrogen bonds that the L-Arg substrate makes with Glu371. Instead, all of these inhibitors induce a conformational change in Glu371, creating an active site with altered molecular recognition properties. The cost of this conformational change is approximately 1-2 kcal, based on our measured constants for inhibitor binding to the wild-type and E371A mutant proteins. These inhibitors derive affinity by pi-stacking above the heme and replacing both intramolecular (Glu371-Met368) and intermolecular (substrate-Trp366) hydrogen bonds to the beta-sheet architecture underlying the active site. When bound to NOS, high-affinity inhibitors in this class are planar, whereas weaker inhibitors are nonplanar. Isozyme differences were observed in the pterin cofactor site, the heme propionate, and inhibitor positions. Computational docking predictions match the crystallographic results, including the Glu371 conformational change and inhibitor-binding orientations, and support a combined crystallographic and computational approach to isozyme-specific NOS inhibitor analysis and design.