Roman Šink

Discovery of Novel 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione Inhibitors of MurD Ligase

We have designed, synthesized, and evaluated 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC(50) in the range 45-206 μM. The high-resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothiazolidin-5-ylidene]methyl)phenylamino}methyl)benzamido)pentanedioic acid [(R)-32] revealed details of the binding mode of the inhibitor within the active site and provides a good foundation for structure-based design of a novel generation of MurD inhibitors.

Design, Synthesis, and Evaluation of New Thiadiazole-Based Direct Inhibitors of Enoyl Acyl Carrier Protein Reductase (InhA) for the Treatment of Tuberculosis

Mycobacterial enoyl acyl carrier protein reductase (InhA) is a clinically validated target for the treatment of tuberculosis infections, a disease that still causes the death of at least a million people annually. A known class of potent, direct, and competitive InhA inhibitors based on a tetracyclic thiadiazole structure has been shown to have in vivo activity in murine models of tuberculosis infection. On the basis of this template, we have here explored the medicinal chemistry of truncated analogues that have only three aromatic rings. In particular, compounds 8b, 8d, 8f, 8l, and 8n show interesting features, including low nanomolar InhA IC50, submicromolar antimycobacterial potency, and improved physicochemical profiles in comparison with the tetracyclic analogues. From this series, 8d is identified as having the best balance of potency and properties, whereby the resolved 8d S-enatiomer shows encouraging in vivo efficacy.

Structure-Based Design of a New Series of D- Glutamic Acid-Based Inhibitors of Bacterial Udp-N-Acetylmuramoyl-L-Alanine:D-Glutamate Ligase (Murd).

MurD ligase is one of the key enzymes participating in the intracellular steps of peptidoglycan biosynthesis and constitutes a viable target in the search for novel antibacterial drugs to combat bacterial drug-resistance. We have designed, synthesized, and evaluated a new series of D-glutamic acid-based Escherichia coli MurD inhibitors incorporating the 5-benzylidenethiazolidin-4-one scaffold. The crystal structure of 16 in the MurD active site has provided a good starting point for the design of structurally optimized inhibitors 73-75 endowed with improved MurD inhibitory potency (IC(50) between 3 and 7 μM). Inhibitors 74 and 75 showed weak activity against Gram-positive Staphylococcus aureus and Enterococcus faecalis. Compounds 73-75, with IC(50) values in the low micromolar range, represent the most potent D-Glu-based MurD inhibitors reported to date.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimers disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimers disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aβ1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215 nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimers disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: Design, synthesis, crystal structures, and biological evaluation

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization.

Synthesis and Biological Evaluation of N‐Acylhydrazones as Inhibitors of MurC and MurD Ligases

The Mur ligases have an essential role in the intracellular biosynthesis of bacterial peptidoglycan, and they represent attractive targets for the design of novel antibacterials. A series of compounds with an N‐acylhydrazone scaffold were synthesized and screened for inhibition of the MurC and MurD enzymes from Escherichia coli. Compounds with micromolar inhibitory activities against both MurC and MurD were identified, and some of them also showed antibacterial activity.

Inhibitors of the peptidoglycan biosynthesis enzymes MurA-F

The widespread emergence of resistant bacterial strains is becoming a serious threat to public health. This thus signifies the need for the development of new antibacterial agents with novel mechanisms of action. Continuous efforts in the design of novel antibacterials remain one of the biggest challenges in drug development. In this respect, the Mur enzymes, MurA-F, that are involved in the formation of UDP-N-acetylmuramyl-pentapeptide can be genuinely considered as promising antibacterial targets. This review provides an in-depth insight into the recent developments in the field of inhibitors of the MurA-F enzymes. Special attention is also given to compounds that act as multiple inhibitors of two, three or more of the Mur enzymes. Moreover, the reasons for the lack of preclinically successful inhibitors and the challenges to overcome these hurdles in the next years are also debated.

Second-generation sulfonamide inhibitors of d-glutamic acid-adding enzyme: Activity optimisation with conformationally rigid analogues of d-glutamic acid

D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities.

Dual Inhibitor of MurD and MurE Ligases from Escherichia coli and Staphylococcus aureus.

MurD and MurE ligases, consecutive enzymes participating in the intracellular steps of bacterial peptidoglycan biosynthesis, are important targets for antibacterial drug discovery. We have designed, synthesized, and evaluated the first d-glutamic acid-containing dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus (IC50 values between 6.4 and 180 μM) possessing antibacterial activity against Gram-positive S. aureus and its methicillin-resistant strain (MRSA) with minimal inhibitory concentration (MIC) values of 8 μg/mL. The inhibitor was also found to be noncytotoxic for human HepG2 cells at concentrations below 200 μM.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.