The enzyme DXS as an anti-infective target:Exploiting multiple hit-identification strategies

We exploited multiple hit-identification strategies toward the development of inhibitors for the enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS). DXS belongs to the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the universal precursors of isoprenoids. The fact that humans exclusively utilize the alternative mevalonate pathway for the synthesis of IDP and DMADP, together with the fact that the MEP pathway is essential in numerous organisms such as Mycobacterium tuberculosis and Plasmodium falciparum, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents. Our projects were supported by computational studies as well as advanced biophysical techniques. Structure-based design and, particularly, fragment-based design were used to model the first fragment-like inhibitors of DXS, for which we validated the binding mode in solution by NMR spectroscopy. By advanced docking studies, we were able to develop and validate a homology model of M. tuberculosis DXS, which can be used for the rational design of inhibitors for M. tuberculosis DXS in the absence of crystallographic information. Ligand-based virtual screening allowed for the identification of the first inhibitors of DXS with double-digit micromolar inhibitory potency and remarkable cell-based activity against multi-drug-resistant strains of M. tuberculosis. The use of the software TPPQuery guided us in the modeling of potential DXS inhibitors that can be conveniently synthesized by multicomponent reactions. We also adopted a simple but powerful hit-identification methodology, namely phage display, for the development of the first peptidic inhibitors of DXS, displaying low micromolar activity against D. radiodurans DXS

Out of the boxes, out of the silos: The need of interdisciplinary collaboration to reduce poor-quality medical products in the supply chain

In this paper, we argue that understanding and addressing the problem of poor-quality medical products requires a more interdisciplinary approach than has been evident to date. While prospective studies based on rigorous standardized methodologies are the gold standard for measuring the prevalence of poor-quality medical products and understanding their distribution nationally and internationally, they should be complemented by social science research to unpack the complex set of social, economic, and governance factors that underlie these patterns. In the following sections, we discuss specific examples of prospective quality surveys and of social science studies, highlighting the value of cross-sector partnerships in driving high-quality, policy-relevant research in this area

How We Treat Drug-Susceptible Pulmonary Tuberculosis: A Practical Guide for Clinicians

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide and pulmonary TB (PTB) is the main variant responsible for fueling transmission of the infection. Effective treatment of drug-susceptible (DS) TB is crucial to avoid the emergence of Mycobacterium tuberculosis-resistant strains. In this narrative review, through a fictional suggestive case of DS PTB, we guide the reader in a step-by-step commentary to provide an updated review of current evidence in the management of TB, from diagnosis to post-treatment follow-up. World Health Organization and Centre for Diseases Control (CDC) guidelines for TB, as well as the updated literature, were used to support this manuscript

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

ShORRT (Short, all-Oral Regimens for Rifampicin-resistant Tuberculosis) Research Package

TDR in close collaboration with the Global TB Programme at WHO and technical partners the WHO Global TB Programme is leading the development of ShORRT (Short, all-Oral Regimens For Rifampicin-resistant Tuberculosis), an operational research package to assess the effectiveness, safety, feasibility, acceptability, cost and impact (including on health-related quality of life) of the use of all-oral shorter drug regimens for adults and children with MDR/RR-TB

Role of prenatal magnetic resonance imaging in fetuses with isolated mild or moderate ventriculomegaly in the era of neurosonography: international multicenter study

Objectives To assess the role of fetal magnetic resonance imaging (MRI) in detecting associated anomalies in fetuses presenting with mild or moderate isolated ventriculomegaly (VM) undergoing multiplanar ultrasound evaluation of the fetal brain. Methods This was a multicenter, retrospective, cohort study involving 15 referral fetal medicine centers in Italy, the UK and Spain. Inclusion criteria were fetuses affected by isolated mild (ventricular atrial diameter, 10.0–11.9 mm) or moderate (ventricular atrial diameter, 12.0–14.9 mm) VM on ultrasound, defined as VM with normal karyotype and no other additional central nervous system (CNS) or extra‐CNS anomalies on ultrasound, undergoing detailed assessment of the fetal brain using a multiplanar approach as suggested by the International Society of Ultrasound in Obstetrics and Gynecology guidelines for the fetal neurosonogram, followed by fetal MRI. The primary outcome of the study was to report the incidence of additional CNS anomalies detected exclusively on prenatal MRI and missed on ultrasound, while the secondary aim was to estimate the incidence of additional anomalies detected exclusively after birth and missed on prenatal imaging (ultrasound and MRI). Subgroup analysis according to gestational age at MRI (< 24 vs ≥ 24 weeks), laterality of VM (unilateral vs bilateral) and severity of dilatation (mild vs moderate VM) were also performed. Results Five hundred and fifty‐six fetuses with a prenatal diagnosis of isolated mild or moderate VM on ultrasound were included in the analysis. Additional structural anomalies were detected on prenatal MRI and missed on ultrasound in 5.4% (95% CI, 3.8–7.6%) of cases. When considering the type of anomaly, supratentorial intracranial hemorrhage was detected on MRI in 26.7% of fetuses, while polymicrogyria and lissencephaly were detected in 20.0% and 13.3% of cases, respectively. Hypoplasia of the corpus callosum was detected on MRI in 6.7% of cases, while dysgenesis was detected in 3.3%. Fetuses with an associated anomaly detected only on MRI were more likely to have moderate than mild VM (60.0% vs 17.7%; P < 0.001), while there was no significant difference in the proportion of cases with bilateral VM between the two groups (P = 0.2). Logistic regression analysis showed that lower maternal body mass index (adjusted odds ratio (aOR), 0.85 (95% CI, 0.7–0.99); P = 0.030), the presence of moderate VM (aOR, 5.8 (95% CI, 2.6–13.4); P < 0.001) and gestational age at MRI ≥ 24 weeks (aOR, 4.1 (95% CI, 1.1–15.3); P = 0.038) were associated independently with the probability of detecting an associated anomaly on MRI. Associated anomalies were detected exclusively at birth and missed on prenatal imaging in 3.8% of cases. Conclusions The incidence of an associated fetal anomaly missed on ultrasound and detected only on fetal MRI in fetuses with isolated mild or moderate VM undergoing neurosonography is lower than that reported previously. The large majority of these anomalies are difficult to detect on ultrasound. The findings from this study support the practice of MRI assessment in every fetus with a prenatal diagnosis of VM, although parents can be reassured of the low risk of an associated anomaly when VM is isolated on neurosonography

Studio della reazione di alfa-arilazione, mediata da palladio, di 4-cromanoni e loro analoghi

Le reazioni di sostituzione di composti carbonilici mediate da complessi di palladio costituiscono una strategia moderna ed altamente efficiente per ottenere l’arilazione o la vinilazione di enolati, spesso anche con eccellente controllo enantiomerico. Tali reazioni tuttavia presentano talvolta problemi di selettività legati alla possibile formazione di prodotti di disostituzione; inoltre, composti carbonilici particolarmente sensibili alle condizioni basiche generalmente impiegate, non possono essere utilizzati. Tra questi vi è il 4-cromanone, il quale subisce l’apertura dell’anello cromanonico in presenza di basi forti. Il nucleo cromanonico è particolarmente interessante in quanto presente in molte sostanze naturali biologicamente attive. Le reazioni di C-3 monoarilazione selettiva di questo substrato costituiscono un’importante via d’accesso ai corrispondenti isoflavanoni, nonché ad un’ampia gamma di strutture più elaborate che da essi derivano. Gli isoflavanoni e i loro derivati rappresentano un importante gruppo di prodotti naturali biologicamente attivi, e alcuni di essi sono inoltre presenti in composti con rilevante attività farmacologica e medicinale. Data la non idoneità dei protocolli di alfa-arilazione classici sul 4-cromanone, abbiamo cercato di individuare opportune condizioni di reazione atte ad ottenere selettivamente la monoarilazione in posizione alfa su questo particolare substrato Inizialmente è stato effettuato un ampio screening delle condizioni di reazione (precursore catalitico, natura della base, solvente) per la messa a punto di un efficiente protocollo che permettesse una selettiva monoarilazione del 4-cromanone ad opera di bromobenzene. Impiegando 2.5 mol % di Pd2(dba)3, 10 mol % di t-Bu3PHBF4, in presenza di 2 equivalenti di KHCO3 come base ed effettuando la reazione in una miscela 4/1 di diossano/H2O a riflusso per 1h 20’ è stato possibile isolare il desiderato 3-fenil-4-cromanone con una resa del 71%. Il rapporto molare ottimale tra 4-cromanone e bromobenzene è risultato pari a 2:1. Le condizioni di reazione messe a punto sono state poi applicate alla C-3 arilazione di 4-cromanoni variamente sostituiti, e a loro analoghi strutturali quali ad esempio il 2-cumaranone ed il 3-isocromanone, ottenendo in particolare per questo ultimo una buona resa nel corrispondente prodotto monoarilato (72%). Come agenti arilanti sono stati impiegati bromuri variamente sostituiti per valutare l’influenza di fattori sterici ed elettronici, tuttavia sembra che i fattori elettronici non giochino un ruolo discriminante nell’efficienza della reazione. E’ interessante inoltre notare che il protocollo messo a punto per la C-3 arilazione diretta del 4-cromanone non soffre di limitazioni dovute all’ingombro sterico sulla porzione arilica: eventuali sostituenti (attivanti e disattivanti) in posizione orto sul bromobenzene, infatti, non ostacolano la reazione permettendo l’isolamento dei desiderati prodotti di C-3 monoarilazione con rese da soddisfacenti a buone (41-72 %). Aumentando la quantità di H2O nella miscela di reazione abbiamo notato che la selettività nei confronti del prodotto monosostituito diminuisce a favore della formazione del prodotto diarilato. Lavorando in largo eccesso di agente arilante (4 equivalenti) e conducendo le reazioni in H2O nelle medesime condizioni ottimizzate per la reazione di monoarilazione, è stato possibile isolare una serie di prodotti di diarilazione (simmetrici e non simmetrici) con rese soddisfacenti. Molte reazioni condotte in H2O sono favorite dalla presenza di tensioattivi capaci di formare micelle in soluzione, le quali permettono la solubilizzazione dei substrati organici grazie all'inclusione di questi nelle cavità lipofile. La porzione idrofila delle micelle, esposta al mezzo acquoso, fa sì che questi "microreattori" siano solubili nel mezzo polare. Alla luce di queste considerazioni, abbiamo provato ad effettuare la reazione di arilazione del 4-cromanone con bromobenzene in H2O in presenza di alcuni tensioattivi, impiegando ancora Pd2(dba)3 / t-Bu3PHBF4 come sistema catalitico e KHCO3 come base. Sono stati testati vari tensioattivi di tipo ionico (Sodio dodecil solfato, esadecil trimetil ammonio bromuro) e non ionico (Polisorbato 80, Poliossietanil-alfa-tocoferil sebacato), impiegandone 5 % (wt) in ogni prova. Dalle prove effettuate è risultato che la selettività di queste reazioni va a favore del prodotto di monosostituzione, al contrario di quanto avveniva in H2O in assenza di tensioattivo. In particolare impiegando poliossietanil-alfa-tocoferil sebacato (PTS) al 5 % (wt) e conducendo la reazione per 2 h a riflusso con un rapporto tra 4-cromanone e bromobenzene pari a 2: 1, è stato possibile isolare il desiderato 3-fenil-4-cromanone con una resa del 70 %. Tuttavia, impiegando agenti arilanti diversi dal bromo benzene (p-bromoanisolo, p-bromofenolo, 3-trifluorometil-bromobenzene), le rese nei corrispondenti composti monosostituiti sono risultate piuttosto basse (31-55 %). Aumentando la percentuale di PTS inserito ed impiegandone il 15 % (wt), invece, sono state ottenute rese molto buone nei prodotti di monoarilazione (67-79 %). Le reazioni condotta con il 15 % (wt) di PTS vanno a completezza in 2 h nel caso in cui si lavori con un rapporto 4-cromanone / bromobenzene pari a 2/1, mentre sono necessarie 24 h nel caso in cui si lavori con 1.2 equivalenti di 4-cromanone per ottenere rese confrontabili (>70 %). Concludendo, abbiamo quindi messo a punto due protocolli per la sintesi di isoflavanoni e analoghi strutturali mediante reazioni di C-3 arilazione diretta di 4-cromanoni e loro analoghi strutturali, substrati su cui questo tipo di reazioni non sono note in letteratura: il primo prevede di effettuare la reazione in opportune condizioni lavorando in diossano/H2O (4/1), mentre il secondo prevede l’impiego di H2O come solvente in presenza di un opportuno tensioattivo capace di formare sistemi micellari in soluzione. Effettuando la reazione in H2O in assenza di tensioattivi, lavorando in eccesso molare dell’agente arilante, è stato possibile inoltre ottenere i corrispondenti derivati alfa, alfa-diarilati seppur con rese piuttosto basse