1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance

1-deoxy-D-xylulose-5-phosphate synthase from <i>Pseudomonas aeruginosa</i> and <i>Klebsiella pneumoniae</i> reveals conformational changes upon cofactor binding

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.</p

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

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Investigating structural insights and inhibitor strategies in MEP pathway enzymes

Drug-resistant infections pose a significant threat to human health with studies predicting they will become the leading cause of death in the next 30 years. There is therefore an urgent need to find new antimicrobial agents. Unfortunately, the antimicrobial drug-discovery and development pipeline is sparse, obscuring hopes of finding new drugs in the near future. The methylerythritol (MEP) pathway used mainly by pathogenic microorganisms has emerged as a source of promising new drug targets for the discovery of anti-infective agents, yet it remains underexplored. This thesis presents a comprehensive study of MEP pathway enzymes with a focus on 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from selected pathogens. We obtained, for the first time, crystal structures of DXPS from Pseudomonas aeruginosa and Klebsiella pneumoniae and explored their structural differences including conformational changes upon binding to the co-factor thiamine diphosphate (ThDP). These findings were instrumental in understanding how the enzyme works and how it could be inhibited. We went on to explore potential inhibition strategies against these enzymes and profiled novel classes of inhibitors, the first was thiamine monophosphate (ThMP) analogues as ThDP competitive inhibitors of DXPS, supported by binding mode analyses by obtaining a co-crystal structure with K. pneumoniae DXPS. Second, we present a promising breakthrough in DXPS inhibition by targeting an allosteric binding site and provide experimental evidence in the form of a co-crystal structure of an allosteric inhibitor with P. aeruginosa DXPS. Furthermore, we analyzed the kinetics and structural variances among IspE kinase homologs. We explored the potential impact of these structural differences on the binding mode of inhibitors, substantiating our findings through co-crystal structures with various inhibitors. This approach offers valuable insights into potential strategies for designing inhibitors. Overall, this work presents novel findings with promising potential to guide antimicrobial drug discovery targeting drug-resistant pathogens and employing underexplored enzyme inhibition approaches.Arzneimittelresistente Infektionen stellen eine erhebliche Bedrohung für die menschliche Gesundheit dar. Studien prognostizieren, dass sie in den nächsten 30 Jahren die führende Todesursache werden könnten. Daher besteht ein dringender Bedarf, neue antimikrobielle Wirkstoffe zu entdecken. Leider ist die Pipeline für die Entdeckung und Entwicklung antimikrobieller Medikamente dünn gesät, was die Hoffnung auf die baldige Entdeckung neuer Medikamente trübt. Der Methylerythritol (MEP)-Weg, der hauptsächlich von pathogenen Mikroorganismen genutzt wird, hat sich als vielversprechende Quelle neuer Wirkstoffziele für die Entdeckung von antiinfektiven Substanzen erwiesen, bleibt jedoch unterexploriert. Diese Dissertation präsentiert eine umfassende Studie zu Enzymen des MEPWegs mit Schwerpunkt auf 1-Deoxy-D-xylulose-5-phosphat-Synthase (DXPS) und 4-Diphosphocytidyl-2-C-methyl-D-erythritol-Kinase (IspE) aus ausgewählten Pathogenen. Zum ersten Mal erhielten wir Kristallstrukturen von DXPS aus Pseudomonas aeruginosa und Klebsiella pneumoniae und erforschten ihre strukturellen Unterschiede, einschließlich konformationeller Veränderungen bei der Bindung an den Cofaktor Thiamindiphosphat (ThDP). Diese Erkenntnisse waren entscheidend, um zu verstehen, wie das Enzym funktioniert und wie es gehemmt werden könnte. Wir untersuchten dann potenzielle Hemmstrategien gegen diese Enzyme und profilten neuartige Klassen von Inhibitoren. Die erste umfasste Thiaminmonophosphat (ThMP)-Analoga als ThDP-konkurrenzfähige Inhibitoren von DXPS, unterstützt durch Bindungsmodusanalysen durch Erlangen einer Co-Kristallstruktur mit K. pneumoniae DXPS. Zweitens präsentieren wir einen vielversprechenden Durchbruch in der Hemmung von DXPS durch die Ausrichtung auf eine allosterische Bindungsstelle und liefern experimentelle Beweise in Form einer Co-Kristallstruktur eines allosterischen Inhibitors mit P. aeruginosa DXPS. Darüber hinaus analysierten wir die Kinetik und strukturellen Unterschiede zwischen IspE-Kinase-Homologen. Wir erforschten das potenzielle Auswirkungen dieser strukturellen Unterschiede auf den Bindungsmodus von Inhibitoren und untermauerten unsere Erkenntnisse durch Co-Kristallstrukturen mit verschiedenen Inhibitoren. Dieser Ansatz bietet wertvolle Einblicke in potenzielle Strategien zur Gestaltung von Inhibitoren. Insgesamt präsentiert diese Arbeit neuartige Erkenntnisse mit vielversprechendem Potenzial, die antimikrobielle Wirkstoffentwicklung zur Bekämpfung von resistenten Pathogenen zu lenken und unterexplorierte Ansätze zur Enzymhemmung zu nutzen

1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritolphosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-Dxylulose-5-phosphate synthase (DXPS) catalyzes a crucial, ratelimiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance

Exploring the Translational Gap of a Novel Class of Escherichia coli IspE Inhibitors

Discovery of novel antibiotics needs multidisciplinary approaches to gain target enzyme and bacterial activities while aiming for selectivity over mammalian cells. Here, we report a multiparameter optimisation of a fragment-like hit that was identified through a structure-based virtual-screening campaign on Escherichia coli IspE crystal structure. Subsequent medicinal-chemistry design resulted in a novel class of E. coli IspE inhibitors, exhibiting activity also against the more pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. While cytotoxicity remains a challenge for the series, it provides new insights on the molecular properties for balancing enzymatic target and bacterial activities simultaneously as well as new starting points for the development of IspE inhibitors with a predicted new mode of action

Evaluation of ketoclomazone and its analogues as inhibitors of 1-deoxy-<scp>d</scp>-xylulose 5-phosphate synthases and other thiamine diphosphate (ThDP)-dependent enzymes

Acknowledgements: We are grateful for the support from K. M. Medhealth (AHYC and TCSH), Research Training Program scholarship from the Australian government (IF), the Canberra Branch of the Australian Red Cross Lifeblood for the provision of red blood cells, members of the van Dooren Lab (Australian National University) for HFF cells, the Schlumberger Foundation Faculty for the Future Fellowship (RH) and the Helmholtz Association Initiative and Networking Fund and the European Research Council, ERC starting grant 757913 (AKHH).Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids.</jats:p

Evaluation of Ketoclomazone and its Analogues as Inhibitors of Thiamine Diphosphate (ThDP)-dependent Enzymes

Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS), a thiamine diphosphate (ThDP)-dependent enzyme, catalyses the first and rate-limiting step of the MEP pathway. Due to its absence in humans, DXPS is considered as an attractive target for the development of anti-infectious agents and herbicides. However, major challenges in designing therapeutic agents that target DXPS include: a) discovering drug-like inhibitors to occupy a highly polar active site; and b) selectively targeting DXPS over other ThDP-dependent enzymes. Ketoclomazone is one of the earliest reported inhibitors of DXPS and antibacterial and herbicidal activities have been documented. This study aimed to investigate the activity of ketoclomazone on DXPS from various species, as well as the broader ThDP-dependent enzyme family. To gain further insights into the inhibition, we have prepared analogues of ketoclomazone and evaluated their activity in biochemical and computational studies. Our findings support the potential of ketoclomazone as a selective antibacterial agent