The synthesis of biologically active heterocycles: development of novel imaging agents for the translocator protein (18 kDa) and poly(ADP-ribose)polymerase 1

The Translocator Protein (18 kDa) (TSPO) resides mainly in microglia and is upregulated in response to neuronal injury and inflammation, rendering it an interesting target for imaging focal neuroinflammation in a range of diseases. A library of TSPO ligands based on PK11195 with positions suitable for [11C]-, [18F]- and [123I]-labelling was prepared using three synthetic approaches. Implementation of a physicochemical study enabled selection of compounds most likely to be brain penetrant, and biological evaluation identified those with high binding affinities for the TSPO. From this, a lead candidate (compound 170) was identified. Radiofluorination and in vitro autoradiography revealed the ability of this compound to image tumour tissue in a mouse model of glioblastoma. Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in the repair of DNA strand breaks, and widely regarded as a therapeutic target for cancer treatment. Many inhibitors of PARP-1 activity exist, e.g. olaparib. A programme of research focussing on the preparation of a potential PET imaging agent for measurement of PARP-1 activity, based on olaparib, was initiated. An expeditious seven step synthetic route was used to prepare a small library of compounds. Preliminary cell-free biological screening of these compounds indicated PARP-1 inhibitory potencies in the low nanomolar range demonstrating potential leads for development of a PET imaging agent, e.g. compound 223. Extracts of Carduus crispus linn., traditionally used in Chinese folk medicine, yield a family of isoquinoline alkaloid natural products (Crispine A–E). Upon commencement of this project no reported synthesis of Crispine C existed in the literature. As such, a facile route utilising a key Pictet-Gams modification of the Bischler-Napieralski reaction for isoquinoline core formation was developed, enabling the first total synthesis of Crispine C to be achieved in seven steps and with an overall product yield of 25%

A novel 18F-labelled high affinity agent for PET imaging of the translocator protein

The translocator protein (TSPO) is an important target for imaging focal neuroinflammation in diseases such as brain cancer, stroke and neurodegeneration, but current tracers for non-invasive imaging of TSPO have important limitations. We present the synthesis and evaluation of a novel 3-fluoromethylquinoline-2-carboxamide, AB5186, which was prepared in eight steps using a one-pot two component indium(III)-catalysed reaction for the rapid and efficient assembly of the 4-phenylquinoline core. Biological assessment and the implementation of a physicochemical study showed AB5186 to have low nanomolar affinity for TSPO, as well as optimal plasma protein binding and membrane permeability properties. Generation of [18F]-AB5186 through 18F incorporation was achieved in good radiochemical yield and subsequent in vitro and ex vivo autoradiography revealed the ability of this compound to bind with specificity to TSPO in mouse glioblastoma xenografts. Initial positron emission tomography imaging of a glioma bearing mouse and a healthy baboon support the potential for [18F]-AB5186 use as a radiotracer for non-invasive TSPO imaging in vivo

Synthesis and evaluation of a radioiodinated tracer with specificity for poly(ADP-ribose) polymerase-1 (PARP-1) in vivo

Interest in nuclear imaging of poly(ADP-ribose) polymerase-1 (PARP-1) has grown in recent years due to the ability of PARP-1 to act as a biomarker for glioblastoma and increased clinical use of PARP-1 inhibitors. This study reports the identification of a lead iodinated analog 5 of the clinical PARP-1 inhibitor olaparib as a potential single-photon emission computed tomography (SPECT) imaging agent. Compound 5 was shown to be a potent PARP-1 inhibitor in cell-free and cellular assays, and it exhibited mouse plasma stability but approximately 3-fold greater intrinsic clearance when compared to olaparib. An (123)I-labeled version of 5 was generated using solid state halogen exchange methodology. Ex vivo biodistribution studies of [(123)I]-5 in mice bearing subcutaneous glioblastoma xenografts revealed that the tracer had the ability to be retained in tumour tissue and bind to PARP-1 with specificity. These findings support further investigations of [(123)I]-5 as a non-invasive PARP-1 SPECT imaging agent

An 18F-labeled poly(ADP-ribose) polymerase positron emission tomography imaging agent

Poly(ADP-ribose) polymerase (PARP) is involved in repair of DNA breaks and is over-expressed in a wide variety of tumors, making PARP an attractive biomarker for positron emission tomography (PET) and single photon emission computed tomography imaging. Consequently, over the past decade, there has been a drive to develop nuclear imaging agents targeting PARP. Here, we report the discovery of a PET tracer that is based on the potent PARP inhibitor olaparib (1). Our lead PET tracer candidate, [18F]20, was synthesized and evaluated as a potential PARP PET radiotracer in mice bearing subcutaneous glioblastoma xenografts using ex vivo biodistribution and PET−magnetic resonance imaging techniques. Results showed that [18F]20 could be produced in a good radioactivity yield and exhibited specific PARP binding allowing visualization of tumors overexpressing PARP. [18F]20 is therefore a potential candidate radiotracer for in vivo PARP PET imaging

Synthesis of the isoquinoline alkaloid, crispine C

The first total synthesis of the isoquinoline alkaloid, crispine C is described in seven steps using a Henry reaction and the Pictet–Gams variant of the Bischler–Napieralski reaction to effect the key transformations

Comparison of Winter Cow Feeding Strategies on Offspring Carcass Characteristics and Meat Quality

Objective The objective of this research was to investigate effects of maternal prepartum dietary energy source (forage vs. concentrate) during mid and late gestation on carcass composition, and meat quality of offspring.Study Description Angus-based cows from 2 sources [n = 129 from SDSU (Experiment 1) and n = 70 from North Dakota State University (Experiment 2)] were stratified by body weight and age and placed into two treatment groups at a drylot facility during mid- and late-gestation: Concentrate (dams fed a concentrate-based diet) or Forage (dams fed a forage-based diet). Calves were finished and carcass data was collected. Striploins were collected for meat quality evaluation

Epidemiology, Public Health, and the Rhetoric of False Positives

BACKGROUND: As an observational science, epidemiology is regarded by some researchers as inherently flawed and open to false results. In a recent paper, Boffetta et al. [Boffetta P, McLaughlin JK, LaVecchia C, Tarone RE, Lipworth L, Blot WJ. False-positive results in cancer epidemiology: a plea for epistemological modesty. J Natl Cancer Inst 100:988-995 (2008)] argued that "epidemiology is particularly prone to the generation of false-positive results." They also said "the tendency to emphasize and over-interpret what appear to be new findings is commonplace, perhaps in part because of a belief that the findings provide information that may ultimately improve public health" and that "this tendency to hype new findings increases the likelihood of downplaying inconsistencies within the data or any lack of concordance with other sources of evidence." The authors supported these serious charges against epidemiology and epidemiologists with few examples. Although we acknowledge that false positives do occur, we view the position of Boffetta and colleagues on false positives as unbalanced and potentially harmful to public health. OBJECTIVE: We aim to provide a more balanced evaluation of epidemiology and its contribution to public health discourse. DISCUSSION: Boffetta and colleagues ignore the fact that false negatives may arise from the very processes that they tout as generating false-positive results. We further disagree with their proposition that false-positive results from a single study will lead to faulty decision making in matters of public health importance. In practice, such public health evaluations are based on all the data available from all relevant disciplines and never to our knowledge on a single study. CONCLUSIONS: The lack of balance by Boffetta and colleagues in their evaluation of the impact of false-positive findings on epidemiology, the charge that "methodological vigilance is often absent" in epidemiologists' interpretation of their own results, and the false characterization of how epidemiologic findings are used in societal decision making all undermine a major source of information regarding disease risks. We reaffirm the importance of epidemiologic evidence as a critical component of the foundation of public health protection

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, <scp>genotype–phenotype</scp> correlations and common mechanisms

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (&gt;60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS‐like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or “DTRs”). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype–phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population