Exploiting multiple hit-identification strategies to identify novel inhibitors of the anti-infective target DXPS

The coronavirus pandemic has raised awareness for infectious diseases, which also put a spotlight on the fight against anti-microbial resistance. A promising new target in this fight is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). Although it has been known for the past few decades, only a few promising inhibitors have been identified so far. For this thesis, multiple hit-identification strategies were pursued with a special focus on Mycobacterium tuberculosis and Plasmodium falciparum DXPS to find new inhibitors. For this thesis a focused fragment library was screened against DXPS in different biophysical assay, in collaboration with the company Atomwise, a virtual screening was performed and three previously identified hit classes were investigated in phenotypic assays against P. falciparum and synthetically optimized. In summary, this thesis has contributed to the identification of several new binders and inhibitors that have promising properties to continue their optimization into leads for drug development.Die Corona Pandemie hat dafür gesorgt, dass Infektionskrankheiten und damit auch der Kampf gegen antibiotikaresistente Keime ins Bewusstsein der Öffentlichkeit gerückt sind. Ein neues Target in diesem Kampf ist die 1-Deoxy-D-xylulose-5-phosphat Synthase (DXPS). Das Enzym ist bereits seit einigen Jahrzehnten bekannt, aber bisher wurden nur wenige Inhibitoren gefunden. In dieser Arbeit wurden verschiedene Hit-Identifikationsstrategien genutzt, um neue Inhibitoren gegen Mycobacterium tuberculosis und Plasmodium falciparum DXPS zu finden. Dafür wurde eine fokussierte Fragmentbibliothek gegen DXPS in verschiedenen biophysikalischen Assays untersucht, ein HPLC-MS/MS-basierter DXPS Assay wurde etabliert, in Kooperation mit der Firma Atomwise wurde ein virtuelles Screening an DXPS durchgeführt und drei bereits bekannte Hit-Klassen wurden im Rahmen dieser Arbeit in phänotypischen Assays gegen P. falciparum getestet und synthetisch optimiert. Zusammengefasst hat diese Arbeit zur Identifikation mehrerer Binder und Inhibitoren beigetragen, die vielversprechende Eigenschaften aufweisen und weiter zu Lead-Verbindungen optimiert und somit für die Medikamentenentwicklung genutzt werden können

Not Every Hit-Identification Technique Works on 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXPS):Making the Most of a Virtual Screening Campaign

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.</p

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Synthesis and structural characterization of new oxorhenium and oxotechnetium complexes with XN2S-tetradentate semi-rigid ligands(X = O, S, N)

Twelve novel oxo-technetium and oxo-rhenium complexes based on N2S2-, N2SO- or N3S-tetradentate semi-rigid ligands have been synthesised and studied herein. By reacting the ligands with a slight excess of suitable [MO]3+ precursor (ReOCl3(PPh3)2 or [NBu4][99gTcOCl4]), the monoanionic complexes of general formula [MO(Ph–XN2S)]− could be easily produced in high yield. The complexes have been characterized by means of IR, electrospray mass spectrometry, elemental analysis, NMR and conductimetry. The crystal structures of [PPh4][ReO(Ph–ON2S)] 1b and [NBu4][99gTcO(Ph–ON2S)] 1c have been established. The [MO]3+ moiety was coordinated via the two deprotonated amide nitrogens, the oxygen and the terminal sulfur atoms in 1b and 1c. In both compounds, the ON2S coordination set is in the equatorial plane, and the complexes adopted a distorted square-pyramidal geometry with an axial oxo-group. The chemical and structural identity of the different prototypic complexes (rhenium, 99gTc complexes and their corresponding 99mTc radiocomplexes) have been also established by a comparative HPLC study

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery