Realising Lead-Oriented Synthesis- A ‘Top-Down’ Approach to Synthesising Lead-Like Scaffolds

This report focusses on the concept of lead-oriented synthesis in an attempt to synthesise diverse small molecule scaffolds that target lead-like chemical space. The ‘top-down’ approach was taken, which is a method used within the Marsden and Nelson group where polycyclic assemblies are prepared and then deconstructed to give a library of diverse lead-like molecules. Each lead-like scaffold was synthesised with good stereo/regio control, on a large scale from cheap starting materials, with appropriate molecular properties. Each compound was then investigated for its diversification at points of the scaffold, in the possibility of providing access to a variety of novel-like, sp3-riched compounds suitable for screening. The computational software LLAMA was used throughout the project to help analyse properties and guide the development of three-dimensional compounds. Chapter 1 discusses the overview of the drug discovery process, detailing the problems faced in the pharmaceutical industry, highlighting the high failure rates for drug candidates possessing certain molecular properties. The ideal molecular properties for drug, leads and fragment compounds is discussed, with the modern synthetic approaches to prepare such lead diverse screening compounds in an efficient manner. Chapter 2 – 4 details the synthesis of diverse novel scaffolds prepared from robust synthetic methodologies to access a library of compounds with appropriate molecular properties for biological screening. In total 14 scaffolds were synthesised from 28 synthetic operations and 52 novel compounds from a total of 73 synthetic operations. Here the method has produced a library of complex and highly three-dimensional compounds with attention paid to physicochemical and functional group properties, whilst maintaining synthetic efficiency. The library was sent for biological evaluation against five different targets, of which 15 hits were observed. A follow-up library was then produced where 13 compounds were sent for a second round of biological evaluation against the Plasmodium falciparum cells

Defining Young in the Context of Prostate Cancer

The experience of prostate cancer is for most men a major life stress with the psychological burden of this disease falling more heavily on those who are younger. Despite this, being young as it applies to prostate cancer is not yet clearly defined with varied chronological approaches applied. However, men’s responses to health crises are closely bound to life course and masculinities from which social roles emerge. This paper applied qualitative methodology (structured focus groups and semistructured interviews with expert informants) using interpretative phenomenological analysis to define what it means to be young and have prostate cancer. Structured focus groups were held with 26 consumer advisors (men diagnosed with prostate cancer who provide support to other men with prostate cancer or raise community awareness) and health professionals. As well, 15 men diagnosed with prostate cancer and in their 40s, 50s, or 60s participated in semi-structured interviews. Participants discussed the attributes that describe a young man with prostate cancer and the experience of being young and diagnosed with prostate cancer. Chronological definitions of a young man were absent or inconsistent. Masculine constructions of what it means to be a young man and life course characteristics appear more relevant to defining young as it applies to prostate cancer compared with chronological age. These findings have implications for better understanding the morbidities associated with this illness, and in designing interventions that are oriented to life course and helping young men reconstruct their identities after prostate cancer

A Carrier for Non-Covalent Delivery of Functional Beta-Galactosidase and Antibodies against Amyloid Plaques and IgM to the Brain

BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words)

A Contraction Stress Model of Hypertrophic Cardiomyopathy due to Sarcomere Mutations.

Thick-filament sarcomere mutations are a common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered four isogenic induced pluripotent stem cell (iPSC) models of β-myosin heavy chain and myosin-binding protein C3 mutations, and studied iPSC-derived cardiomyocytes in cardiac microtissue assays that resemble cardiac architecture and biomechanics. All HCM mutations resulted in hypercontractility with prolonged relaxation kinetics in proportion to mutation pathogenicity, but not changes in calcium handling. RNA sequencing and expression studies of HCM models identified p53 activation, oxidative stress, and cytotoxicity induced by metabolic stress that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as a direct consequence of thick-filament mutations, irrespective of mutation localization, and the p53 pathway as a molecular marker of contraction stress and candidate therapeutic target for HCM patients

Defining young in the context of prostate cancer

The experience of prostate cancer is for most men a major life stress with the psychological burden of this disease falling more heavily on those who are younger. Despite this, being young as it applies to prostate cancer is not yet clearly defined with varied chronological approaches applied. However, men’s responses to health crises are closely bound to life course and masculinities from which social roles emerge. This paper applied qualitative methodology (structured focus groups and semistructured interviews with expert informants) using interpretative phenomenological analysis to define what it means to be young and have prostate cancer. Structured focus groups were held with 26 consumer advisors (men diagnosed with prostate cancer who provide support to other men with prostate cancer or raise community awareness) and health professionals. As well, 15 men diagnosed with prostate cancer and in their 40s, 50s, or 60s participated in semi-structured interviews. Participants discussed the attributes that describe a young man with prostate cancer and the experience of being young and diagnosed with prostate cancer. Chronological definitions of a young man were absent or inconsistent. Masculine constructions of what it means to be a young man and life course characteristics appear more relevant to defining young as it applies to prostate cancer compared with chronological age. These findings have implications for better understanding the morbidities associated with this illness, and in designing interventions that are oriented to life course and helping young men reconstruct their identities after prostate cancer

A longitudinal study of muscle strength and function in patients with cancer cachexia

Purpose Patients with cancer frequently experience an involuntary loss of weight (in particular loss of muscle mass), defined as cachexia, with profound implications for independence and quality of life. The rate at which such patients’ physical performance declines has not been well established. The aim of this study was to determine the change in muscle strength and function over 8 weeks in patients with already established cancer cachexia, to help inform the design and duration of physical activity interventions applicable to this patient group. Methods Patients with thoracic and gastrointestinal cancer, with unintentional weight loss of >5% in 6 months or BMI < 20 plus 2% weight loss were included. Physical and functional assessments (baseline, 4 weeks, 8 weeks) included: isometric quadriceps and hamstring strength, handgrip, standing balance, 10m walk time and timed up and go. Results Fifty patients (32 male), mean ±SD age 65 ±10 years and BMI 24.9 ±4.3kg/m2 were recruited. Thoracic cancer patients had lower muscle strength and function (p0.05). Baseline variables did not differentiate between completers and non-completers (p>0.05). Conclusions More than a third of patients with established cancer cachexia in our study were stable over 8 weeks, suggesting a subgroup who may benefit from targeted interventions of reasonable duration. Better understanding the physical performance parameters which characterize and differentiate these patients has important clinical implications for cancer multidisciplinary team practice

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.United States. National Institutes of Health (1R01GM104948)United States. National Institutes of Health (1DP1NS087724)United States. National Institutes of Health ( NIH 1R01EY023173)United States. National Institutes of Health (1U01MH106011