Ternary complex formation of AFN‐1252 with Acinetobacter baumannii FabI and NADH: Crystallographic and biochemical studies

Acinetobacter baumannii is an opportunistic Gram‐negative bacterial pathogen, associated mostly with hospital‐acquired infections. The emergence of drug resistance strains made it necessary to explore new pathways for the development of more effective antibiotics. Enoyl CoA reductase (FabI), a key enzyme in the fatty acid biosynthesis (FAS) pathway, has emerged as a potential target for antibacterial drug development. Earlier reports show that the lead SaFabI inhibitor AFN‐1252 can inhibit FabI from other organisms including Escherichia coli and Burkholderia pseudomallei, but with differential potency. In the present work, we show that AFN‐1252 is a moderate inhibitor of AbFabI with an IC₅₀ of 216 nM. AFN‐1252 stabilized AbFabI with a 4.2°C increase in the melting temperature (T_m) and, interestingly, the stabilization effect was significantly increased in presence of the cofactor NADH (?T_m = 17°C), suggesting the formation of a ternary complex AbFabI: AFN‐1252: NADH. X‐ray crystallography studies of AbFabI co‐crystalized with AFN‐1252 and NADH confirmed the ternary complex formation. The critical interactions of AFN‐1252 with AbFabI and NADH identified from the co‐crystal structure may facilitate the design and development of new drugs against A. baumannii infections by targeting the FAS pathway.     

Novel agents for the treatment of resistant Gram-positive infections

Bacteria have proved themselves able to develop resistance to every antibiotic used clinically. Traditional agents used for treatment of serious infections caused by Gram-positive species have recently been supplemented with the introduction of linezolid, quinupristin-dalfopristin, several new quinolones and telithromycin. However, resistance to many of these agents has already been reported and, although each currently retains activity against the vast majority of clinical isolates of its target species, their long-term efficacy is uncertain. We must look to develop other compounds to replace and hopefully improve upon existing anti-Gram-positive agents. Daptomycin (a lipopeptide), oritavancin and dalbavancin (both second-generation glycopeptides) and ramoplanin (a glycolipodepsipeptide) are among the agents in advanced stages of development and, at present, many seem likely to proceed to licensing. In addition, it is encouraging that many agents active against novel bacterial targets have been discovered and are in earlier stages of development. In the next two decades, we should be optimistic that a regular flow of new anti-Gram-positive agents will enable us to offset the constant spectre of bacterial resistance.     

Analysis of Enoyl‐Acyl Carrier Protein Reductase Structure and Interactions Yields an Efficient Virtual Screening Approach and Suggests a Potential Allosteric Site

Enoyl‐acyl carrier protein reductases have an important role in fatty acid biosynthesis and are considered essential for bacterial and protozoal survival. Here, we perform a computational assessment of enoyl‐acyl carrier protein reductase structures, providing insights for inhibitor design that we incorporate into a virtual screening approach. Firstly, we analyse 80 crystal structures of 16 different enoyl‐acyl carrier protein reductases for their active site characteristics and druggability, finding these sites contain a readily druggable pocket, of varying size and shape. Interestingly, a high affinity, potentially allosteric site was identified for pfFabI. Analysis of the ligand–protein interactions of four enoyl‐acyl carrier protein reductases from different micro‐organisms (InhA, pfFabI, saFabI and ecFabI), involving 59 available crystal structures, found three commonly shared interactions; constraining these interactions in docking improved enrichment of enoyl‐acyl carrier protein reductase virtual screens, by up to 60% in the top 3% of the ranked library. This docking protocol also improved pose prediction, decreasing the root‐mean‐square deviation to crystallographic pose by up to 75% on average. The binding site analysis and knowledge‐based docking protocol presented here can potentially assist in the structure‐based design of new enoyl‐acyl carrier protein reductase inhibitors.     

Safety,tolerability and pharmacokinetics of multiple oral doses of AFN-1252 administered as immediate release (IR) tablets in healthy subjects

Background: AFN-1252 is a novel inhibitor of FabI, which is essential in Staphylococcus spp. This study evaluated the safety, tolerability and pharmacokinetics of multiple oral doses of AFN-1252 immediate-release tablets.

Methods: Part I evaluated AFN-1252 as a single 200 mg dose in fed versus fasted subjects. Part II evaluated 200, 300 and 400 mg doses of AFN-1252 administered once-daily for 10 days.

Results: Pharmacokinetics indicated good absorption with a median T_max of 2–3 hours, and a mean t_1/2 of 7–10 hours, for all doses. C_max and AUC responses were non-linear. A high-fat meal reduced AUC_0–t and C_max values by 62% and 48%, respectively, and delayed T_max by 2.5 hours. All adverse events, including possibly drug-related headache and nausea, were mild or moderate.

Conclusions: Multiple ascending doses of AFN-1252 were safe and well tolerated. AFN-1252 has potential for once- or twice-daily dosing in the treatment of staphylococcal infections.     

Structure‐based drug design and in vitro testing reveal new inhibitors of enoyl‐acyl carrier protein reductases

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl‐acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro‐organisms. In this project, we conducted structure‐based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC₅₀ values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC₅₀ value of 7.8 μM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.     

New drug-targeting strategy from beneath the shell of egg

Importance of the field: Antibiotic resistance is a serious problem that continues to challenge the healthcare sectors and has become increasingly alarming in the past few years. To face this emerging global crisis, there is a need to find a new class of antibiotics that act on new microbial targets and/or harness existing antibiotics by developing new drug-targeting strategies.

Areas covered in this review: This review: explores an innovative drug-delivery strategy of using hen egg lysozyme as a carrier to enable water solubilization and to allow specific targeting to the microbial cells of a water-insoluble antimicrobial agent with a powerful killing action; addresses potentials for lysozyme in antibiotics drug targeting; and provides insight for the future direction of this highly prospective technology.

What the reader will gain: The unique features and advantages of lysozyme-based drug delivery system are highlighted. The efficiency of lysozyme in solubilization and delivery of lipophilic antibiotics, to reformulate drugs that may fail clinical trials owing to low solubility, is emphasized.

Take home message: Fewer pharmaceutical companies are inventing new antibiotics because of long development times and high failure rates. Combining lysozyme with a powerful old antibiotic may open doors to revolutionizing medicine, particularly in the treatment of deadly infections.     

Evaluation of antiprotozoal and plasmodial enoyl-ACP reductase inhibition potential of turkish medicinal plants

A total of 58 extracts of different polarity were prepared from various organs of 16 species of Turkish plants and screened for their antitrypanosomal, antileishmanial and antiplasmodial activities. No significant activity was observed against Trypanosoma cruzi, whereas many extracts showed appreciable trypanocidal potential against T. brucei rhodesiense, with the CHCl(3)-soluble portion of Phlomis kurdica being the most active (IC(50) 2.7 microg[sol ]mL). Almost all extracts, particularly the CHCl(3) phases, exhibited growth inhibition activity against Leishmania donovani amastigotes. The CHCl(3)-solubles of Putoria calabrica roots (IC(50) 1.9 microg[sol ]mL), Wendlandia ligustroides leaves (IC(50) 2.1 microg[sol ]mL) and Rhododendron luteum leaves (IC(50) 2.3 microg[sol ]mL) displayed the highest leishmanicidal potential. The majority of the extracts also possessed antiplasmodial activity against the multi-drug resistant K1 Plasmodium falciparum strain. The most potent antiplasmodial activity was observed with the CHCl(3) extracts of Phlomis kurdica (IC(50) 1.5 microg[sol ]mL), P. leucophracta (IC(50) 1.6 microg[sol ]mL), Scrophularia cryptophila (IC(50) 1.8 microg[sol ]mL), Morina persica (IC(50) 1.9 microg[sol ]mL) and the aqueous root extract of Asperula nitida subsp. subcapitellata (IC(50) 1.6 microg[sol ]mL). Twenty-one extracts with significant antimalarial activity (IC(50) < 5 microg[sol ]mL) were also tested for their ability to inhibit the purified enoyl-ACP reductase (FabI), a crucial enzyme in the fatty acid biosynthesis of P. falciparum. The CHCl(3) extract of Rhododendron ungernii leaves (IC(50) 10 microg[sol ]mL) and the H(2)O-soluble portion of Rhododendron smirnovii leaves (IC(50) 0.4 microg[sol ]mL) strongly inhibited the FabI enzyme. The preliminary data indicate that some (poly)phenolic compounds are responsible for the FabI inhibition potential of these extracts. The presented work reports for the first time the antiprotozoal activity of nine different genera as well as a target specific antimalarial screening for the identification of P. falciparum FabI inhibitors from medicinal plant extracts.     

Enoyl acyl carrier protein reductase inhibitors: an updated patent review (2011 – 2015)

Introduction: Enoyl-(acyl-carrier-protein) reductase (ENR) is a limiting step enzyme in the Fatty Acid Synthase II system. In mammals, there is no homologue to ENR, which makes it an optimal candidate target for selective anti-infective drugs. Up-to-date, only two ENR inhibitors are used in clinical practice.

Area covered: This review is a survey on important patents on low molecular weight compounds with ENR inhibiting activity published in 2011–2015. Common patent databases (SciFinder, esp@cenet, WIPO) were used to locate patent applications on the proposed topic and in the timespan of 2011–2015.

Expert opinion: In 2011–2015, we have observed patents in previously known structural groups of diphenyl ethers and acrylamides as well as new structural classes, often identified by high-throughput screening campaigns. The spectrum of activity of applied derivatives covers significant bacteria, mycobacteria, and apicomplexan parasites (Plasmodia and Toxoplasma). Good news from research of ENR inhibitors: a) four selective anti-staphylococcal compounds applied in 2011–2015 or earlier were pushed to Phase I or Phase II clinical trials and some of them proved safety and tolerability after peroral and/or intravenous administration; b) big pharma companies have renewed their interest in the development of new anti-infective compounds against resistant strains of clinical relevance.     

Design,Synthesis, Structural Characterization by IR, 1H, 13C, 15N, 2D‐NMR,X‐Ray Diffraction and Evaluation of a New Class of Phenylaminoacetic Acid Benzylidene Hydrazines as pfENR Inhibitors

Recent studies have revealed that plasmodial enoyl‐ACP reductase (pfENR, FabI), one of the crucial enzymes in the plasmodial type II fatty acid synthesis II (FAS II) pathway, is a promising target for liver stage malaria infections. Hence, pfENR inhibitors have the potential to be used as causal malarial prophylactic agents. In this study, we report the design, synthesis, structural characterization and evaluation of a new class of pfENR inhibitors. The search for inhibitors began with a virtual screen of the iResearch database by molecular docking. Hits obtained from the virtual screen were ranked according to their Glide score. One hit was selected as a lead and modified to improve its binding to pfENR; from this, a series of phenylamino acetic acid benzylidene hydrazides were designed and synthesized. These molecules were thoroughly characterized by IR, ¹H, ¹³C, ¹⁵N, 2D‐NMR (COSY, NOESY, ¹H‐¹³C, ¹H‐¹⁵N HSQC and HMBC), and X‐ray diffraction. NMR studies revealed the existence of conformational/configurational isomers around the amide and imine functionalities. The major species in DMSO solution is the E, E form, which is in dynamic equilibrium with the Z, E isomer. In the solid state, the molecule has a completely extended conformation and forms helical structures that are stabilized by strong hydrogen bond interactions, forming a helical structure stabilized by N‐H…O interactions, a feature unique to this class of compounds. Furthermore, detailed investigation of the NMR spectra indicated the presence of a minor impurity in most compounds. The structure of this impurity was deduced as an imidazoline‐4‐one derivative based on ¹H‐¹³C and ¹H‐¹⁵H HMBC spectra and was confirmed from the NOESY spectra. The molecules were screened for in vitro activity against recombinant pfENR enzyme by a spectrophotometric assay. Four molecules, viz. 17, 7, 10, and 12 were found to be active at 7, 8, 10, and 12 μm concentration, respectively, showing promising pfENR inhibitory potential. A classification model was derived based on a binary QSAR approach termed recursive partitioning (RP) to highlight structural characteristics that could be tuned to improve activity.