17 research outputs found
Kinetic template-guided tethering of fragments
This thesis is composed of two separate projects: Kinetic Template-Guided Tethering of Fragments and Design and
Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after
DNA Damage.
Kinetic Template-Guided Tethering of Fragments
The development of a novel methodology for the site-directed discovery of small molecule, protein-binding ligands is
described. The protein of interest, with a cysteine thiol (either native or engineered) adjacent to the desired binding
pocket, is incubated with mixtures of low molecular weight compounds (fragments) modified with either an
acrylamide or a vinyl sulfonamide capture group. Any ligand within the mixture that binds within the pocket brings
the capture group into close proximity with the cysteine thiol, promoting a conjugate addition reaction at an increased
rate over the background reaction. The capture reaction is designed to be slow, such that during the time course of an
experiment, no adduct formation is observed unless the reaction is templated by the protein. By this method, binding
ligands are rapidly identified by mass spectrometry analysis of the crude reaction mixtures. The methodology has
been termed ‘kinetic template-guided tethering’. Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1
Degradation after DNA Damage
An inhibitor described within the literature was found to attenuate the reduction of cyclin D1 after DNA damage to
cells. In order to implement a two-step chemical proteomics strategy to find the molecular target of this inhibitor, a
synthesis of the compound with an alkyne appendage was required. The alkyne acts as a functional handle for
attachment of a reporter molecule in cells via the Huisgen cycloaddition (‘Click’) reaction. The design and synthesis
of this inhibitor with the alkyne appendage is described.Open Acces
Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease
COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease
FP tethering: a screening technique to rapidly identify compounds that disrupt protein–protein interactions
High-throughput kinetic analysis for target-directed covalent ligand discovery
Cysteine‐reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high‐quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan‐reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine‐reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2‐selective allosteric (type IV) kinase inhibitor whose novel mode‐of‐action could be exploited therapeutically
Intrinsic reactivity profile of electrophilic moieties to guide covalent drug design: N-α-acetyl- l
A major outbreak of asthma associated with a thunderstorm: Experience of accident and emergency departments and patients' characteristics
Objective - To investigate the time course of an epidemic of asthma after a thunderstorm, characteristics of patients affected, and the demand on emergency medical resources. Design - Study of registers and records in accident and emergency departments and questionnaire to staff Setting - London area. Subjects - All patients presenting at 12 accident and emergency departments with asthma or other airways disease. Main outcome measures-Numbers of patients, clinical features, information on shortage of resources-equipment, drugs, and staff. Results - The epidemic had a sudden onset on 24 June 1994; 640 patients with asthma or other airways disease attended during 30 hours from 1800 on 24 June, nearly 10 times the expected number. Over half (365) the patients were aged 21 to 30 years. A history of hay fever was recorded in 403 patients; for 283 patients this was the first known attack of asthma; a history of chronic obstructive airways disease was recorded in 12 patients. In all, 104 patients were admitted (including five to an intensive care unit). Several departments ran out of equipment or drugs, caned in additional doctors, or both. Concusions - This study supports the view that this epidemic was larger than previously reported epidemics and the hypothesis that 'thunderstorm associated asthma' is related to aeroallergens. Demands on resources were considerable; a larger proportion of patients needing intensive care would have caused greater problems
A major outbreak of asthma associated with a thunderstorm: Experience of accident and emergency departments and patients' characteristics
Objective - To investigate the time course of an epidemic of asthma after a thunderstorm, characteristics of patients affected, and the demand on emergency medical resources. Design - Study of registers and records in accident and emergency departments and questionnaire to staff Setting - London area. Subjects - All patients presenting at 12 accident and emergency departments with asthma or other airways disease. Main outcome measures-Numbers of patients, clinical features, information on shortage of resources-equipment, drugs, and staff. Results - The epidemic had a sudden onset on 24 June 1994; 640 patients with asthma or other airways disease attended during 30 hours from 1800 on 24 June, nearly 10 times the expected number. Over half (365) the patients were aged 21 to 30 years. A history of hay fever was recorded in 403 patients; for 283 patients this was the first known attack of asthma; a history of chronic obstructive airways disease was recorded in 12 patients. In all, 104 patients were admitted (including five to an intensive care unit). Several departments ran out of equipment or drugs, caned in additional doctors, or both. Concusions - This study supports the view that this epidemic was larger than previously reported epidemics and the hypothesis that 'thunderstorm associated asthma' is related to aeroallergens. Demands on resources were considerable; a larger proportion of patients needing intensive care would have caused greater problems