The hit-and-return system enables efficient time-resolved serial synchrotron crystallography.

We present a 'hit-and-return' (HARE) method for time-resolved serial synchrotron crystallography with time resolution from milliseconds to seconds or longer. Timing delays are set mechanically, using the regular pattern in fixed-target crystallography chips and a translation stage system. Optical pump-probe experiments to capture intermediate structures of fluoroacetate dehalogenase binding to its ligand demonstrated that data can be collected at short (30 ms), medium (752 ms) and long (2,052 ms) intervals.

Molecular recognition in chemical and biological systems.

Structure-based ligand design in medicinal chemistry and crop protection relies on the identification and quantification of weak noncovalent interactions and understanding the role of water. Small-molecule and protein structural database searches are important tools to retrieve existing knowledge. Thermodynamic profiling, combined with X-ray structural and computational studies, is the key to elucidate the energetics of the replacement of water by ligands. Biological receptor sites vary greatly in shape, conformational dynamics, and polarity, and require different ligand-design strategies, as shown for various case studies. Interactions between dipoles have become a central theme of molecular recognition. Orthogonal interactions, halogen bonding, and amide⋅⋅⋅π stacking provide new tools for innovative lead optimization. The combination of synthetic models and biological complexation studies is required to gather reliable information on weak noncovalent interactions and the role of water.

Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid.

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.

A dinuclear zinc(II) complex of a new unsymmetric ligand with an N(5)O(2) donor set: a structural and functional model for the active site of zinc phosphoesterases.

The dinuclear complex [Zn(2)(DPCPMP)(pivalate)](ClO4), where DPCPMP is the new unsymmetrical ligand [2-(N-(3-((bis((pyridin-2-yl)methyl)amino)methyl)-2-hydroxy-5-methylbenzyl)-N-((pyridin-2-yl)methyl)amino)acetic acid], has been synthesized and characterized. The complex is a functional model for zinc phosphoesterases with dinuclear active sites. The hydrolytic efficacy of the complex has been investigated using bis-(2,4-dinitrophenyl)phosphate (BDNPP), a DNA analog, as substrate. Speciation studies using potentiometric titrations have been performed for both the ligand and the corresponding dizinc complex to elucidate the formation of the active hydrolysis catalyst; they reveals that the dinuclear zinc(II) complexes, [Zn(2)(DPCPMP)](2+) and [Zn(2)(DPCPMP)(OH)](+) predominate the solution above pH4. The relatively high pK(a) of 8.38 for water deprotonation suggests that a terminal hydroxide complex is formed. Kinetic investigations of BDNPP hydrolysis over the pH range 5.5-11.0 and with varying metal to ligand ratio (metal salt:ligand=0.5:1 to 3:1) have been performed. Variable temperature studies gave the activation parameters ΔH(‡)=95.6kJmol(-1), ΔS(‡)=-44.8Jmol(-1)K(-1), and ΔG(‡)=108.0 kJmol(-1). The cumulative results indicate the hydroxido-bridged dinuclear Zn(II) complex [Zn(2)(DPCPMP)(µ-OH)](+) as the effective catalyst. The mechanism of hydrolysis has been probed by computational modeling using density functional theory (DFT). Calculations show that the reaction goes through one concerted step (S(N)2 type) in which the bridging hydroxide in the transition state becomes terminal and performs a nucleophilic attack on the BDNPP phosphorus; the leaving group dissociates simultaneously in an overall inner sphere type activation. The calculated free energy barrier is in good agreement with the experimentally determined activation parameters.

Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.