72,586 research outputs found
Crystallographic fragment screening - improvement of workflow, tools and procedures, and application for the development of enzyme and protein-protein interaction modulators
One of the great societal challenges of today is the fight against diseases which reduce
life expectancy and lead to high economic losses. Both the understanding and the
addressing of these diseases need research activities at all levels. One aspect of this is
the discovery and development of tool compounds and drugs. Tool compounds support
disease research and the development of drugs. For about 20 years, the discovery of new
compounds has been attempted by screening small organic molecules by high-throughput
methods. More recently, X-ray crystallography has emerged as the most promising method
to conduct such screening. Crystallographic fragment-screening (CFS) generates binding
information as well as 3D-structural information of the target protein in complex with the
bound fragment. This doctoral research project is focused primarily on the optimization of
the crystallographic fragment screening workflow. Investigated were the requirements for
more successful screening campaigns with respect to the crystal system studied, the
fragment libraries, the handling of the crystalline samples, as well as the handling of the
data associated with a screening campaign. The improved CFS workflow was presented
as a detailed protocol and as an accompanying video to train future CFS users in a
streamlined and accessible way. Together, these improvements make CFS campaigns a
more high-throughput method, offering the ability to screen larger fragment libraries and
allowing higher numbers of campaigns performed per year. The protein targets throughout
the project were two enzymes and a spliceosomal protein-protein complex. The enzymes
comprised the aspartic protease Endothiapepsin and the SARS-Cov-2 main protease. The
protein-protein complex was the RNaseH-like domain of Prp8, a vital structural protein in
the spliceosome, together with its nuclear shuttling factor Aar2. By performing the CFS
campaigns against disease-relevant targets, the resulting fragment hits could be used
directly to develop tool compounds or drugs. The first steps of optimization of fragment
hits into higher affinity binders were also investigated for improvements. In summary, a
plethora of novel starting points for tool compound and drug development was identified
Biophysical and Biochemical Screening Approaches for Antimicrobial Drug Discovery Targeting S. aureus ClpP
The discovery of antibacterial drugs has been among most significant achievements of mankind in saving millions of lives across the planet from infectious diseases. With rise in resistance to almost all existing chemotypes, the design of next generation novel antibiotics has become much more challenging and difficult. The early 21st century witnessed the advancement of multiple novel chemotypes during golden age of antibiotics however the pace of antibiotic drug discovery has slowed down tremendously, contributing to life threatening antimicrobial discovery void since 1980’s. Therefore the need to develop novel antibiotics with unique mechanism of action to leverage against multi drug resistance pathogens, is paramount. In this direction the Caseinolytic Protease P (ClpP) is an emerging drug discovery target with significant potential for treatment of recalcitrant biofilm forming infections from pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) This dissertation highlights the ongoing efforts to facilitate the discovery of novel non peptidic ClpP activator compounds and improvement of pharmacological profile of existing ClpP targeting Acyldepsipeptides (ADEPs) series antibiotics. The chapter one discusses the history and synopsis of conventional antibiotics drug discovery screening approaches, and transitions to modern era structure or fragment based screening approaches. The merits and challenges of such approaches of targeting a well conserved bacterial protease (ClpP) are discussed along with dissertation aims toward development of biophysical and biochemical screening approaches. Chapter two discusses optimization of thermal shift assay as primary screening assay for ClpP and its utility toward screening of fragment collections and buffer conditions. Chapter three discussed the development of a site specific Fluorescence Polarization based FP probe based on ADEP scaffold and its utility as a robust high throughput capable primary screening assay for screening of diverse collections ranging from bioactives to fragments. Chapter four discusses development of a label free Surface Plasmon Resonance (SPR) based assay geared toward screening of fragment as well as in house small and large (ADEP analogs) series compounds in addition to determining full kinetics for lead prioritization. Chapter five discusses the results of multiple screening campaigns utilizing combination of above assays to generate multiple hits with superior ligand efficiency and chemical tractability. Chapter six concludes with analysis of the best of compounds among individual series or from screening campaigns and highlights effectiveness of above screening assays toward hit exploration along with outlook on anticipated challenges and future directions
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Studies within Fragment-Based Drug Discovery: Library Synthesis and Hit-to-Lead Optimisation
This thesis reports two projects aimed at addressing challenges within fragment-based drug discovery.
The first project describes efforts towards utilising synthetic methodology to address deficiencies within fragment screening collections. This involved the development of a modular, robust and scalable route to access α,α-disubstituted amino ester building blocks, which in turn were derivatised to allow the rapid assembly of five (a total of eight in collaboration) spirocyclic scaffolds. Importantly, this library was structurally diverse, comprising three (a total of six in collaboration) pharmacophore-like heterocycles and carbocycles. Moreover, numerous three-dimensional exit vectors were incorporated within each core spirocycle, and the ability of these handles to effect a diverse set of chemical modifications was exemplified through the generation of 16 (a total of 21 in collaboration) examples. Computational studies highlighted the excellent physicochemical and 3D properties of the library, as well as the broad coverage of underexplored chemical space that was achieved. This library was also screened for antibacterial activity in a phenotypic assay against the clinically relevant bacterial strains, Pseudomonas aeruginosa and Staphylococcus aureus.
The second project examined the inhibition of propionate detoxification mechanisms in bacteria as an attractive strategy for the development of antibacterial agents. A fragment screening campaign against 2-methylcitrate synthase (PrpC) from Pseudomonas aeruginosa identified several hit compounds based on an indole unit. Synthetic efforts were undertaken to elaborate these fragment hits to increase potency. The adopted strategy focused on growing the indole fragment towards the nearby oxaloacetate binding pocket and occupying it with a fragment mimicking its natural substrate. This approach yielded a compound with an in vitro half maximal inhibitory concentration of 130 µM against the enzymatic activity of PrpC
Structure-Guided Evolution of Potent and Selective CHK1 Inhibitors through Scaffold Morphing
Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice
Virtual screening for inhibitors of the human TSLP:TSLPR interaction
The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Ra), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP: TSLPR complex to investigate the drugability of this complex. Two commercially available fragment libraries were screened computationally for possible inhibitors and a selection of fragments was subsequently tested in vitro. The screening setup consisted of two orthogonal assays measuring TSLP binding to TSLPR: a BLI-based assay and a biochemical assay based on a TSLP: alkaline phosphatase fusion protein. Four fragments pertaining to diverse chemical classes were identified to reduce TSLP: TSLPR complex formation to less than 75% in millimolar concentrations. We have used unbiased molecular dynamics simulations to develop a Markov state model that characterized the binding pathway of the most interesting compound. This work provides a proof-ofprinciple for use of fragments in the inhibition of TSLP: TSLPR complexation
The Effective Fragment Molecular Orbital Method for Fragments Connected by Covalent Bonds
We extend the effective fragment molecular orbital method (EFMO) into
treating fragments connected by covalent bonds. The accuracy of EFMO is
compared to FMO and conventional ab initio electronic structure methods for
polypeptides including proteins. Errors in energy for RHF and MP2 are within 2
kcal/mol for neutral polypeptides and 6 kcal/mol for charged polypeptides
similar to FMO but obtained two to five times faster. For proteins, the errors
are also within a few kcal/mol of the FMO results. We developed both the RHF
and MP2 gradient for EFMO. Compared to ab initio, the EFMO optimized structures
had an RMSD of 0.40 and 0.44 {\AA} for RHF and MP2, respectively.Comment: Revised manuscrip
Pharmacogenomics in children: advantages and challenges of next generation sequencing applications
Pharmacogenetics is considered as a prime example of how personalized medicine nowadays can be put into practice. However, genotyping to guide pharmacological treatment is relatively uncommon in the routine clinical practice. Several reasons can be found why the application of pharmacogenetics is less than initially anticipated, which include the contradictory results obtained for certain variants and the lack of guidelines for clinical implementation. However, more reproducible results are being generated, and efforts have been made to establish working groups focussing on evidence-based clinical guidelines. For another pharmacogenetic hurdle, the speed by which a pharmacogenetic profile for a certain drug can be obtained in an individual patient, there has been a revolution in molecular genetics through the introduction of next generation sequencing (NGS), making it possible to sequence a large number of genes up to the complete genome in a single reaction. Besides the enthusiasm due to the tremendous increase of our sequencing capacities, several considerations need to be made regarding quality and interpretation of the sequence data as well as ethical aspects of this technology. This paper will focus on the different NGS applications that may be useful for pharmacogenomics in children and the challenges that they bring on
Terminal restriction fragment length polymorphism is an “old school” reliable technique for swift microbial community screening in anaerobic digestion
The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants
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