This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 μg/mL). Lack of activity against E. coli was maintained (IC50 > 20 μM and MIC > 128 μg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein.

Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l‐phenylalanine (l‐Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l‐Phe is the key player in this transition. X‐ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr138‐loop to the active site in the presence of l‐Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr138 point mutants. A high linear correlation (r2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate‐activated full‐length tetramers. In the tetramers, a correlation (r2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l‐Phe concentration. The new data support a similar functional importance of the Tyr138‐loop in the catalytic domain and the full‐length enzyme homotetramer.

Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 μM inhibitor is described herein.

The ATPase subunit of DNA gyrase B is an attractive antibacterial target due to high conservation across bacteria and the essential role it plays in DNA replication. A novel class of pyrazolopyridone inhibitors was discovered by optimizing a fragment screening hit scaffold using structure guided design. These inhibitors show potent Gram-positive antibacterial activity and low resistance incidence against clinically important pathogens.

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

Chitinases of the GH18 family play important roles in a variety of pathogenic organisms and have also been shown to be involved in human asthma progression, making these enzymes potential drug targets. While a number of potent GH18 chitinase inhibitors have been described, in general, these compounds suffer from limited synthetic accessibility or unfavorable medicinal-chemical properties, making them poor starting points for the development of chitinase-targeted drugs. Exploiting available structural data, we have rationally designed bisdionin C, a submicromolar inhibitor of GH18 enzymes, that possesses desirable druglike properties and tractable chemical synthesis. A crystallographic structure of a chitinase-bisdionin C complex shows the two aromatic systems of the ligand interacting with two conserved tryptophan residues exposed in the active site cleft of the enzyme, while at the same time forming extensive hydrogen-bonding interactions with the catalytic machinery. The observed mode of binding, together with inhibition data, suggests that bisdionin C presents an attractive starting point for the development of specific inhibitors of bacterial-type, but not plant-type, GH 18 chitinases.

Acidic mammalian chitinase (AMCase) is produced in the lung during allergic inflammation and asthma, and inhibition of enzymatic activity has been considered as a therapeutic strategy. However, most chitinase inhibitors are nonselective, additionally inhibiting chitotriosidase activity. Here, we describe bisdionin F, a competitive AMCase inhibitor with 20-fold selectivity for AMCase over chitotriosidase, designed by utilizing the AMCase crystal structure and dicaffeine scaffold. In a murine model of allergic inflammation, bisdionin F-treatment attenuated chitinase activity and alleviated the primary features of allergic inflammation including eosinophilia. However, selective AMCase inhibition by bisdionin F also caused dramatic and unexpected neutrophilia in the lungs. This class of inhibitor will be a powerful tool to dissect the functions of mammalian chitinases in disease and represents a synthetically accessible scaffold to optimize inhibitory properties in terms of airway inflammation.

Protein crystallography has proven to be an effective method of obtaining high-resolution structures of protein-ligand complexes. However, in certain cases only apoprotein structures are readily available and the generation of crystal complexes is more problematic. Some crystallographic systems are not amenable to soaking of ligands owing to crystal-packing effects and many protein-ligand complexes do not crystallize under the same conditions as used for the apoprotein. Using crystals of human phosphodiesterase 10a (hPDE10a) as an example of such a challenging crystallographic system, the structure of the complex with papaverine was obtained to 2.8 A resolution using protein crystals cross-linked by glutaraldehyde prior to soaking of the ligand. Inspection of the electron-density maps suggested that the correct mode of binding was obtained in one of the two monomers in the asymmetric unit and inspection of crystal-packing contacts explained why cocrystallization experiments and soaking of crystals that were not cross-linked were unsuccessful.

Fragment-based drug discovery (FBDD) represents a change in strategy from the screening of molecules with higher molecular weights and physical properties more akin to fully drug-like compounds, to the screening of smaller, less complex molecules. This is because it has been recognised that fragment hit molecules can be efficiently grown and optimised into leads, particularly after the binding mode to the target protein has been first determined by 3D structural elucidation, e.g. by NMR or X-ray crystallography. Several studies have shown that medicinal chemistry optimisation of an already drug-like hit or lead compound can result in a final compound with too high molecular weight and lipophilicity. The evolution of a lower molecular weight fragment hit therefore represents an attractive alternative approach to optimisation as it allows better control of compound properties. Computational chemistry can play an important role both prior to a fragment screen, in producing a target focussed fragment library, and post-screening in the evolution of a drug-like molecule from a fragment hit, both with and without the available fragment-target co-complex structure. We will review many of the current developments in the area and illustrate with some recent examples from successful FBDD discovery projects that we have conducted.

A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp residue to the solid support and assembly of the linear peptide chain by Fmoc SPPS prior to cyclisation and side-chain manipulation on-resin. Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection. A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure. The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides, and antiasthmatics. Argifin, a natural product cyclopentapeptide, competitively inhibits family 18 chitinases in the nanomolar to micromolar range and shows extensive substrate mimicry. In an attempt to map the active fragments of this large natural product, the cyclopentapeptide was progressively dissected down to four linear peptides and dimethylguanylurea, synthesized using a combination of solution and solid phase peptide synthesis. The peptide fragments inhibit chitinase B1 from Aspergillus fumigatus (AfChiB1), the human chitotriosidase, and chitinase activity in lung homogenates from a murine model of chronic asthma, with potencies ranging from high nanomolar to high micromolar inhibition. X-ray crystallographic analysis of the chitinase-inhibitor complexes revealed that the conformations of the linear peptides were remarkably similar to that of the natural product. Strikingly, the dimethylguanylurea fragment, representing only a quarter of the natural product mass, was found to harbor all significant interactions with the protein and binds with unusually high efficiency. The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.

Chitinases catalyse the hydrolysis of chitin, the natural homopolymer of β(1,4)-linked N-acetyl-D-glucosamine. Chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. Chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. In this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. The argifi n structure includes two sensitive β-linked Asp residues, as well as an unusual carbamoylated Arg side chain, while argadin contains a unique Asp β- semialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. We will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [1,2] This is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. The application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described.

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central β-sheet enclosed by α-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.