Initial Experiences with Amyloid-Related Imaging Abnormalities in Patients Receiving Aducanumab Following Accelerated Approval.

Aducanumab is the first FDA-approved amyloid-lowering immunotherapy for Alzheimer's disease. There is little real-world data to guide management of amyloid-related imaging abnormalities (ARIA), a potentially serious side-effect which requires surveillance with magnetic resonance imaging. We report our experiences in managing ARIA in patients receiving aducanumab at the Butler Hospital Memory and Aging Program during the year following FDA approval. We followed the Appropriate Use Recommendations for aducanumab to guide patient selection, detection, and management of ARIA (1). ARIA-E occurred in 6 out of 24 participants treated; all APOE-ε4 carriers. Treatment was discontinued in 4 cases of moderate-severe ARIA-E, temporarily held in 1 moderate case, and dosed through in 1 mild case (mean duration = 3 months, range, 1-6 months). No participants required hospitalization or high dose corticosteroids. Participants on anticoagulation were excluded and no macrohemorrhages occurred. These data support the measured approaches to treatment outlined in the Appropriate Use Recommendations.

Synthesis and Evaluation of Cereblon-Recruiting HaloPROTACs.

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

Evaluation of 1,2,3-Triazoles as Amide Bioisosteres In Cystic Fibrosis Transmembrane Conductance Regulator Modulators VX-770 and VX-809.

The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11, derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

Mirror-Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide.

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Šresolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Šand by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.

Analysis of risk factors for elbow dysplasia in giant breed dogs.

OBJECTIVE: Identify radiographic risk factors for development of elbow dysplasia in giant breed dogs less than one year of age. METHODS: Twenty-five giant breed puppies (Bernese Mountain dogs, English Mastiff, and Newfoundland) were studied. Both elbows of each dog were radiographed monthly from two to six months of age, then every other month until radial and ulnar physeal closure, followed two months later by bilateral elbow computed tomography. Radiographic parameters measured included the presence or absence of a separate centre of ossification of the anconeal process (SCOAP), medial coronoid disease (MCD), ununited anconeal process, humeral osteochondrosis, elbow incongruity, as well as the length of the radius and ulna, radius-to-ulna ratio, and date of closure of the radial and ulnar physes. RESULTS: Fifteen dogs completed the study. Two Bernese Mountain dogs were diagnosed with MCD. Risk factors significantly associated with medial coronoid disease included dyssynchronous physeal closure and a decreased radius-to-ulna ratio, both detected between eight to 11 months of age. A separate centre of ossification of the anconeal process was present in 60% of the dogs, and was not a risk factor for development of elbow dysplasia. CLINICAL SIGNIFICANCE: Transient, dyssynchronous growth of the radius and ulna may be a risk factor for development of MCD in Bernese Mountain dogs. Dyssynchronous physeal closure or decreased radius-to-ulna ratio prior to radiographic closure of the distal ulnar and radial physes warrants further study in Bernese Mountain dogs and other breeds subject to MCD development.

Crystal Structures Reveal that the Reaction Mechanism of Imidazoleglycerol-Phosphate Dehydratase Is Controlled by Switching Mn(II) Coordination.

Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.

Enantioselective cyclization of enamide-ynes and application to the synthesis of the kopsifoline core.

We report the palladium-catalyzed enantioselective cyclization of 1,6-enamidynes to form spirocyclic ring systems. We applied this methodology to the concise synthesis of the skeletal core of the kopsifoline alkaloids.

Breast cancer, side population cells and ABCG2 expression.

Recurrent metastatic breast cancer may arise in part due to the presence of drug resistant adult stem cells such as Side Population (SP) cells, whose phenotype has been demonstrated to be due to the expression of ABCG2. We hypothesised that SP may be identified in Fine Needle Aspirates (FNAs) and their presence may be determined by expression of ABCG2 in breast tumours. SP and non-side population cells (NSP) were isolated using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux and analysed for expression of ABCG2 and chemoresistance. FNA samples used in SP analysis were matched with paraffin-embedded tissue which was used in immunohistochemical analysis to assess ABCG2 expression. Results were correlated to the pathobiology of the tumour. MCF7 and MDA-MB-231 cell lines contain SP cells. MCF7 SP have increased expression of ABCG2 and increased resistance to mitoxantrone compared to NSP cells. The presence of SP in FNAs were significantly associated with ER-negative (p=0.008) and with triple negative breast cancers (p=0.011) which were also found to have a significant increase in ABCG2 protein expression. ABCG2 transcript was detected in some but not all SP cell populations isolated from FNA samples.

Enantioselective cyclizations of silyloxyenynes catalyzed by cationic metal phosphine complexes.

The discovery of complementary methods for enantioselective transition metal-catalyzed cyclization with silyloxyenynes has been accomplished using chiral phosphine ligands. Under palladium catalysis, 1,6-silyloxyenynes bearing a terminal alkyne led to the desired five-membered ring with high enantioselectivities (up to 91% ee). As for reactions under cationic gold catalysis, 1,6- and 1,5-silyloxyenynes bearing an internal alkyne furnished the chiral cyclopentane derivatives with excellent enantiomeric excess (up to 94% ee). Modification of the substrate by incorporating an α,ß-unsaturation led to the discovery of a tandem cyclization. Remarkably, using silyloxy-1,3-dien-7-ynes under gold catalysis conditions provided the bicyclic derivatives with excellent diastereo- and enantioselectivities (up to >20:1 dr and 99% ee).

An investigation into infant feeding in children born with a cleft lip and/or palate in the West of Scotland.

AIM: To investigate feeding practices in infants born with a cleft lip and/or palate (CLP) in the West of Scotland and the challenges that the parents of these children experienced especially in the first hours and months after birth. METHODS: A questionnaire involving a 'face-to-face' interview was completed with parents of cleft children under the age of 6 years with a cleft lip and/or palate attending the Oral Orthopaedic Prevention Clinic (OOPC). RESULTS: 90 questionnaires were completed and analysed. The incidence of breastfeeding in this study at birth was 54%. In comparison, the incidence of breastfeeding in Scotland nationally was 63% in the year 2000 and 70% in 2005 indicating a lower uptake of breastfeeding for this CLP population. Cleft type had a significant impact on whether the infant was breastfed (p<0.05), those with a cleft lip being more likely to be breastfed. Twenty-nine percent of cleft infants required the use of a naso-gastric tube (NGT) to assist feeding either in hospital during the days following birth or later when there were concerns about the infant's weight. Of these all but one had a CP+/- CL, (p<0.001); 26% of parents reported that their infant had used a pre-surgical appliance; 70% rated the appliance highly in terms of aiding feeding. The help and support given by the cleft team, especially Specialist Cleft Nurses (SCNs), was rated as positive in over 95% of cases but was less positive for the non-cleft health care professionals. Parents found it difficult to find the right feeding method for their baby until they received input from the SCNs and only a minority of parents managed to establish a regular feeding pattern. CONCLUSIONS: This study recommends the employment of more SCNs and an improvement of the knowledge of non-cleft health care professionals.

Dental caries prevalence in children with cleft lip/palate aged between 6 months and 6 years in the West of Scotland.

AIM: To compare the prevalence of dental caries in children with cleft lip/palate with national data at the same age. METHOD: Ethical approval was granted from the West of Scotland Ethics Committee. Children attending the Oral Orthopaedic Clinic were examined for caries according to the criteria of the British Association for the Study of Community Dentistry (BASCD) by two trained and calibrated examiners (KB, RW). Subjects were divided into five age groups: 0.5-1.49; 1.5-2.49; 2.5-3.49; 3.5-4.49 and 4.5-6.0 years. Mean dmft scores were compared with available national data (National Dental Inspection Program of Scotland) for nursery [NDIP 3-year-old survey 2008, unpublished] and 5-year-old children in Scotland [NDIP, 2008]. RESULTS: 209 subjects were examined (participation rate of 87.4%); 45.9% were female, 54.1% male; 21 children (10%) had a recognised syndrome and were reported separately. Cleft Palate (CP) was the most commonly occurring cleft in both syndromic and non-syndromic groups, followed in decreasing numbers by Unilateral Cleft Lip and Palate (UCLP), Bilateral Cleft Lip and Palate (BCLP), Unilateral Cleft Lip (UCL) and Bilateral Cleft Lip (BCL). The only age group with a significantly higher level of dental caries compared with national data was the 4.5-6.0 year-olds where only 37.2% of the children with clefts were caries free compared with the national figure of 57.7% (p=0.004). This same age group had a mean dmft for the cleft group of 3.24 compared with 1.86 nationally. The other age groups had similar dmft and percentages of subjects caries free compared with the national data. The differences did not reach significance. Caries was more common in the anterior teeth of the youngest two age groups, but in the posterior teeth of the two oldest age groups. CONCLUSION: By the age of 4.5 years, children with cleft lip/palate in the West of Scotland have significantly more caries than their non-cleft peers.

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily.

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.

99mTc-interleukin-2 scintigraphy in normal subjects and in patients with autoimmune thyroid diseases: a feasibility study.

PURPOSE: Radiolabelled interleukin-2 is a radiopharmaceutical used for the study of chronic inflammatory processes. (123)I-labelled interleukin-2 has successfully been used in a large number of patients affected by several immune-mediated diseases. (123)I, however, is expensive and not readily available. We have, therefore, developed a method for labelling interleukin-2 with (99m)Tc to high specific activity based on the use of an N(3)S bifunctional chelating agent. In this paper, we describe the results obtained with (99m)Tc-interleukin-2 in a series of eight normal subjects and of 12 patients with autoimmune thyroid diseases. METHODS: Biodistribution, pharmacokinetics, haematological and systemic toxicity, radiation absorbed dose and in vivo targeting were studied. RESULTS: Results showed rapid plasma clearance of (99m)Tc-interleukin-2 with retention mainly in the kidneys. Biodistribution and kinetics were similar to that observed for (123)I-interleukin-2. No acute systemic toxicity was found; a small decrease in peripheral blood lymphocytes was observed in the first hours only in patients, but it was mild and transient. (99m)Tc-interleukin-2 accumulated, to varying extents, in the thyroid of all patients affected by autoimmune thyroid diseases but not in the thyroid of normal subjects. The effective dose equivalent of a diagnostic activity of (99m)Tc-interleukin-2 (185 MBq) was 1.35 mSv. No correlation was observed between thyroid autoantibodies and uptake of (99m)Tc-interleukin-2. CONCLUSIONS: The use of (99m)Tc-interleukin-2 is safe and simple; the favourable dosimetry and biodistribution and the rapid clearance make it potentially useful for the study of chronic inflammatory diseases such as autoimmune thyroid disease.

Differential diagnosis of adnexal masses: risk of malignancy index, ultrasonography, magnetic resonance imaging, and radioimmunoscintigraphy.

A risk of malignancy index (RMI), based on menopausal status, ultrasound (US) findings, and serum CA125, has previously been described and validated in the primary evaluation of women with adnexal masses and is widely used in selective referral of women from local cancer units to specialized cancer centers. Additional imaging modalities could be useful for further characterization of adnexal masses in this group of women. A prospective cohort study was conducted of 196 women with an adnexal mass referred to a teaching hospital for diagnosis and management. Follow-up data was obtained for 180 women; 119 women had benign and 61 women malignant adnexal masses. The sensitivity and specificity of specialist US, magnetic resonance imaging (MRI), radioimmunoscintigraphy (RS), and the RMI were determined. We identified a subgroup of women with RMI values of 25-1000 where the value of further specialist imaging was evaluated. Sensitivity and specificity for specialist US were 100% and 57%, for MRI 92% and 86%, and for RS 76% and 87%, respectively. Analysis of 123 patients managed sequentially, using RMI cutoff values of > or =25 and <1000 and then US and MRI provided a sensitivity of 94% and a specificity of 90%. Using this RMI cutoff followed by specialist US and MRI, as opposed to the traditional RMI cutoff value of 250, can increase the proportion of patients with cancer appropriately referred in to a cancer center, with no change in the proportion of patients with benign disease being managed in a local unit.

Selective parathyroid venous sampling in patients with complicated hyperparathyroidism.

OBJECTIVE: The role of preoperative localisation of abnormal parathyroid glands remains controversial but is particularly relevant to the management of patients with recurrent or persistent hyperparathyroidism and familial syndromes. We report our experience of the use of selective parathyroid venous sampling (PVS) in the localisation of parathyroid disease in such patients. DESIGN: We report a retrospective 10-year experience (n = 27) of the use of PVS in complicated primary hyperparathyroidism and contrast the use of PVS with neck ultrasound, magnetic resonance imaging (MRI), computed tomography (CT) and sestamibi imaging modalities. RESULTS: In 14 out of 25 patients who underwent surgery PVS results were completely concordant with surgical and histological findings and 88% of patients achieved post-operative cure. Out of 13 patients referred after previous failed surgery, 12 underwent further surgery which was curative in 9. In total PVS yielded useful positive (n = 13) and/or negative information (n = 6) in 19 out of 25 patients undergoing surgery. Using histology as the gold standard, 59% of PVS studies were entirely consistent with histology, as compared with 39% of ultrasound scans, 36% of sestamibi scans and 17% of MRI/CT scans. CONCLUSIONS: PVS is a valuable adjunct to MRI/CT and sestamibi scanning in selected patients with complicated hyperparathyroidism when performed in an experienced unit.

Analysis of protein solvent interactions in glucose dehydrogenase from the extreme halophile Haloferax mediterranei.

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-A resolution under crystallization conditions which closely mimic the "in vivo" intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.