The GCKR-P446L gene variant predisposes to raised blood cholesterol and lower blood glucose in the P446L mouse-a model for GCKR rs1260326.

OBJECTIVES: The Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L. METHODS: GKRP-deficient hepatocytes were transfected with adenoviral vectors for human or mouse GKRP:446 P or 446 L for cellular comprehensive analysis including transcriptomics consequent to P446 > L. Physiological traits in the diet-challenged P446L mouse were compared with pleiotropic associations at the human rs1260326 locus. Transcriptomics was compared in P446L mouse liver with hepatocytes overexpressing glucokinase or GKRP:446 P/L. RESULTS: 1. P446 > L substitution in mouse or human GKRP similarly compromises protein expressivity of GKRP:446 L, nuclear sequestration of glucokinase and counter-regulation of gene expression. 2. The P446L knockin mouse has lower liver glucokinase and GKRP protein similar to human liver homozygous for rs1260326-446 L. 3. The diet-challenged P446L mouse has lower blood glucose, raised blood cholesterol and altered hepatic cholesterol homeostasis consistent with relative glucokinase-to-GKRP excess, but not raised blood triglycerides. CONCLUSIONS: Mouse GKRP phenocopies the human GKRP:P446 > L substitution despite the higher affinity for glucokinase of human GKRP. The diet-challenged P446L mouse replicates several traits found in association with the rs1260326 locus on chromosome 2 including raised blood cholesterol, lower blood glucose and lower liver glucokinase and GKRP protein but not raised blood triglycerides.

Effect of AZD4017, a Selective 11ß-HSD1 Inhibitor, on Bone Turnover Markers in Postmenopausal Osteopenia.

CONTEXT: The causative link between circulating glucocorticoid excess and osteoporosis is well-established. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which increases local cortisol production, is expressed in human osteoblasts and its activity increases with age. OBJECTIVE: We hypothesized that local 11ß-HSD1 might mediate an age-related decrease in bone formation and that selective 11ß-HSD1 inhibition may enhance bone formation. METHODS: A dual-center, phase II, randomized, double-blind, placebo-controlled trial of 90 days' treatment with AZD4017 (a selective 11ß-HSD1 inhibitor) was conducted in 55 postmenopausal women with osteopenia. Participants received 400 mg oral AZD4017 twice daily vs matched placebo over 90 days. The primary outcome measure was the impact on the bone formation marker osteocalcin. Secondary objectives included correlation with 11ß-HSD1 activity. RESULTS: At 90 days, osteocalcin levels did not differ between treatment groups: active (mean 22.3 [SD 8.6] ng/mL, n = 22) and placebo (21.7 [SD 9.2] ng/mL, n = 24), with a baseline-adjusted treatment effect of 0.95 (95% CI: -2.69, 4.60). The results from the urinary [THF + alloTHF]/THE ratio (index of 11ß-HSD1 activity) and the urinary cortisol/cortisone ratio (index of 11ß-HSD2 activity) confirmed a > 90% inhibition of 11ß-HSD1 but no change in activity of 11ß-HSD2. CONCLUSION: This trial demonstrates that AZD4017 selectively inhibits 11ß-HSD1 activity in vivo in a safe and reversible manner. Following 90 days of treatment, there is no effect on bone formation, indicating that the relative impairment of bone mineral density in postmenopausal women is not mediated by local intracellular production of cortisol under normal physiological concentrations.

Discovery and mechanism of action studies of 4,6-diphenylpyrimidine-2-carbohydrazides as utrophin modulators for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a fatal disease with no cure, caused by lack of the cytoskeletal protein dystrophin. Upregulation of utrophin, a dystrophin paralogue, offers a potential therapy independent of mutation type. The failure of first-in-class utrophin modulator ezutromid/SMT C1100 in Phase II clinical trials necessitates development of compounds with better efficacy, physicochemical and ADME properties and/or complementary mechanisms. We have discovered and performed a preliminary optimisation of a novel class of utrophin modulators using an improved phenotypic screen, where reporter expression is derived from the full genomic context of the utrophin promoter. We further demonstrate through target deconvolution studies, including expression analysis and chemical proteomics, that this compound series operates via a novel mechanism of action, distinct from that of ezutromid.

Public-Private Partnerships: Compound and Data Sharing in Drug Discovery and Development.

Collaborative efforts between public and private entities such as academic institutions, governments, and pharmaceutical companies form an integral part of scientific research, and notable instances of such initiatives have been created within the life science community. Several examples of alliances exist with the broad goal of collaborating toward scientific advancement and improved public welfare. Such collaborations can be essential in catalyzing breaking areas of science within high-risk or global public health strategies that may have otherwise not progressed. A common term used to describe these alliances is public-private partnership (PPP). This review discusses different aspects of such partnerships in drug discovery/development and provides example applications as well as successful case studies. Specific areas that are covered include PPPs for sharing compounds at various phases of the drug discovery process-from compound collections for hit identification to sharing clinical candidates. Instances of PPPs to support better data integration and build better machine learning models are also discussed. The review also provides examples of PPPs that address the gap in knowledge or resources among involved parties and advance drug discovery, especially in disease areas with unfulfilled and/or social needs, like neurological disorders, cancer, and neglected and rare diseases.

11ßHSD1 Inhibition with AZD4017 Improves Lipid Profiles and Lean Muscle Mass in Idiopathic Intracranial Hypertension.

BACKGROUND: The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) determines prereceptor metabolism and activation of glucocorticoids within peripheral tissues. Its dysregulation has been implicated in a wide array of metabolic diseases, leading to the development of selective 11ß-HSD1 inhibitors. We examined the impact of the reversible competitive 11ß-HSD1 inhibitor, AZD4017, on the metabolic profile in an overweight female cohort with idiopathic intracranial hypertension (IIH). METHODS: We conducted a UK multicenter phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017. Serum markers of glucose homeostasis, lipid metabolism, renal and hepatic function, inflammation and androgen profiles were determined and examined in relation to changes in fat and lean mass by dual-energy X-ray absorptiometry. RESULTS: Patients receiving AZD4017 showed significant improvements in lipid profiles (decreased cholesterol, increased high-density lipoprotein [HDL] and cholesterol/HDL ratio), markers of hepatic function (decreased alkaline phosphatase and gamma-glutamyl transferase), and increased lean muscle mass (1.8%, P < .001). No changes in body mass index, fat mass, and markers of glucose metabolism or inflammation were observed. Patients receiving AZD4017 demonstrated increased levels of circulating androgens, positively correlated with changes in total lean muscle mass. CONCLUSIONS: These beneficial metabolic changes represent a reduction in risk factors associated with raised intracranial pressure and represent further beneficial therapeutic outcomes of 11ß-HSD1 inhibition by AZD4017 in this overweight IIH cohort. In particular, beneficial changes in lean muscle mass associated with AZD4017 may reflect new applications for this nature of inhibitor in the management of conditions such as sarcopenia.

11ß-Hydroxysteroid dehydrogenase type 1 inhibition in idiopathic intracranial hypertension: a double-blind randomized controlled trial.

Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability and investigated indicators of in vivo efficacy of the 11ß-hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared with placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017 or placebo was conducted. Women aged 18-55 years with active idiopathic intracranial hypertension (>25 cmH2O lumbar puncture opening pressure and active papilledema) were included. Participants received 400 mg of oral AZD4017 twice daily compared with matching placebo over 12 weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropometric measures. In vivo efficacy was evaluated in the central nervous system and systemically. A total of 31 subjects [mean age 31.2 (SD = 6.9) years and body mass index 39.2 (SD = 12.6) kg/m2] were randomized to AZD4017 (n = 17) or placebo (n = 14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH2O) compared with placebo (31.3 cmH2O), but the difference between groups was not statistically significant (mean difference: -2.8, 95% confidence interval: -7.1 to 1.5; P = 0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group [mean change: -4.3 cmH2O (SD = 5.7); P = 0.009] but not in the placebo group [mean change: -0.3 cmH2O (SD = 5.9); P = 0.8]. AZD4017 was safe, with no withdrawals related to adverse effects. Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11ß-hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation, serum and cerebrospinal fluid cortisol:cortisone ratios) demonstrated significant enzyme inhibition with the reduction in serum cortisol:cortisone ratio correlating significantly with reduction in lumbar puncture pressure (P = 0.005, R = 0.70). This is the first phase II randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe and well tolerated and inhibited 11ß-hydroxysteroid dehydrogenase type 1 activity in vivo. Reduction in serum cortisol:cortisone correlated with decreased intracranial pressure. Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest.

Chronic glucokinase activator treatment activates liver Carbohydrate response element binding protein and improves hepatocyte ATP homeostasis during substrate challenge.

AIM: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. METHODS: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. RESULTS: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-ß), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-ß mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. CONCLUSIONS: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-ß and both catalytic subunits of AMP-activated protein kinase.

Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches.

Duchenne muscular dystrophy (DMD) is a devastating progressive disease for which there is currently no effective treatment except palliative therapy. There are several promising genetic approaches, including viral delivery of the missing dystrophin gene, read-through of translation stop codons, exon skipping to restore the reading frame and increased expression of the compensatory utrophin gene. The lessons learned from these approaches will be applicable to many other disorders.

Pharmacologically targeting the primary defect and downstream pathology in Duchenne muscular dystrophy.

DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-ß, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.

Progress in therapy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a devastating muscular dystrophy of childhood. Mutations in the dystrophin gene destroy the link between the internal muscle filaments and the extracellular matrix, resulting in severe muscle weakness and progressive muscle wasting. There is currently no cure and, whilst palliative treatment has improved, affected boys are normally confined to a wheelchair by 12 years of age and die from respiratory or cardiac complications in their twenties or thirties. Therapies currently being developed include mutation-specific treatments, DNA- and cell-based therapies, and drugs which aim to modulate cellular pathways or gene expression. This review aims to provide an overview of the different therapeutic approaches aimed at reconstructing the dystrophin-associated protein complex, including restoration of dystrophin expression and upregulation of the functional homologue, utrophin.

Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle. There is significant evidence demonstrating that increasing levels of the dystrophin-related protein, utrophin, in mouse models results in sarcolemmal bound utrophin and prevents the muscular dystrophy pathology. The aim of this work was to develop a small molecule which increases the levels of utrophin in muscle and thus has therapeutic potential. METHODOLOGY AND PRINCIPAL FINDINGS: We describe the in vivo activity of SMT C1100; the first orally bioavailable small molecule utrophin upregulator. Once-a-day daily-dosing with SMT C1100 reduces a number of the pathological effects of dystrophin deficiency. Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise; a surrogate for the six minute walk test currently recommended as the pivotal outcome measure in human trials for DMD. CONCLUSIONS AND SIGNIFICANCE: This study demonstrates proof-of-principle for the use of in vitro screening methods in allowing identification of pharmacological agents for utrophin transcriptional upregulation. The best compound identified, SMT C1100, demonstrated significant disease modifying effects in DMD models. Our data warrant the full evaluation of this compound in clinical trials in DMD patients.

Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2.

Heterozygous mutations of GATA3, which encodes a dual zinc-finger transcription factor, cause hypoparathyroidism with sensorineural deafness and renal dysplasia. Here, we have investigated the role of GATA3 in parathyroid function by challenging Gata3+/- mice with a diet low in calcium and vitamin D so as to expose any defects in parathyroid function. This led to a higher mortality among Gata3+/- mice compared with Gata3+/+ mice. Compared with their wild-type littermates, Gata3+/- mice had lower plasma concentrations of calcium and parathyroid hormone (PTH) and smaller parathyroid glands with a reduced Ki-67 proliferation rate. At E11.5, Gata3+/- embryos had smaller parathyroid-thymus primordia with fewer cells expressing the parathyroid-specific gene glial cells missing 2 (Gcm2), the homolog of human GCMB. In contrast, E11.5 Gata3-/- embryos had no Gcm2 expression and by E12.5 had gross defects in the third and fourth pharyngeal pouches, including absent parathyroid-thymus primordia. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays showed that GATA3 binds specifically to a functional double-GATA motif within the GCMB promoter. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and, with GCM2/B, forms part of a transcriptional cascade in parathyroid development and function.

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions.

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca(2+), demonstrated its Mn(2+)-transporting activity, showed its Ca(2+)- and Mn(2+)-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of (45)Ca(2+) (K(0.5) = 0.27 microm) or (54)Mn(2+) transport into an A23187-releasable compartment.

Hailey-Hailey disease: identification of novel mutations in ATP2C1 and effect of missense mutation A528P on protein expression levels.

ATP2C1, encoding the human secretory pathway Ca(2+)-ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey disease (HHD), an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis. In this study, we used denaturing high-performance liquid chromatography to screen all 28 exons and flanking intron boundaries of ATP2C1 for mutations in 9 HHD patients. Nine different mutations were identified. Five of these mutations, including one nonsense, one deletion, two splice-site, and one missense mutation, have not been previously reported. Recently, functional analysis of a series of site-specific mutants, designed to mimic missense mutations found in ATP2C1, uncovered specific defects in Ca(2+) and/or Mn(2+) transport and protein expression in mutant hSPCA1 polypeptides. In order to investigate the molecular and physiological basis of HHD in the patient carrying missense mutation A528P, located in the putative nucleotide binding domain of the molecule, site-directed mutagenesis was employed to introduce this mutation into the wild-type ATP2C1 (hSPCA1) sequence. Functional analyses of HHD-mutant A528P demonstrated a low level of protein expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. Analogous to conclusions drawn from our previous studies, these results further support the theory of haploinsufficiency as a prevalent mechanism for the dominant inheritance of HHD, by suggesting that the level of hSPCA1 in epidermal cells is critical.

Effect of Hailey-Hailey Disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1).

ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1 approximately P --> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.

The replication and stable-inheritance functions of IncP-9 plasmid pM3.

Little is known of the transfer and maintenance machinery of the IncP-9 plasmids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to streptomycin and tetracycline, was found repeatedly in Pseudomonas species from numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 degrees C and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding Km(R). The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptide products were found to have similarity to previously analysed proteins involved in plasmid replication (rep gene), transfer (mpf; mating-pair formation gene) and stable maintenance (par, mrs genes). The organization of these genes showed similarity to several plasmid systems including the Ti and pSYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the region required for replication, and identified the putative rep gene and putative par promoter region as able to express incompatibility. rep deletion mutants were lost from the cell line, and expression of the rep gene was shown to be controlled by negative autoregulation. A pMT2 derivative with an insertion between the rep and par genes showed very weak, if any, ability to replicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.