Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans─The Role of a New Putative Membrane-Associated Domain and Proton-Relay System in Catalysis.

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.

ß-Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies.

ß-Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to ß-lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography were used to understand the mechanisms of BLG-PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80-fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the ß-barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

N-Edited Guanine Isosteres.

Guanine is one out of five endogenous nucleobases and of key interest in drug discovery and chemical biology. Hitherto, the synthesis of guanine derivatives involves lengthy multistep sequential synthesis of low overall diversity, resulting in the quest for innovation. Using a "single-atom skeletal editing" approach, we designed 2-aminoimidazo[2,1-f][1,2,4]triazin-4(3H)-one as a guanine isostere, conserving the biologically important HBA-HBD-HBD (HBA = hydrogen bond acceptor; HBD = hydrogen bond donor) substructure. We realized our design by a simple one-pot two-step method combining the Groebke-Blackburn-Bienaymé reaction (GBB-3CR) and a deprotection reaction to assemble the innovative guanine isosteres in moderate to good yields. Our innovative, diverse, short, and reliable multicomponent reaction synthesis will add to the toolbox of guanine isostere syntheses.

Highly Stereoselective Ugi/Pictet-Spengler Sequence.

Discovering novel synthetic routes for rigid nitrogen-containing polyheterocycles using sustainable, atom-economical, and efficient (= short) synthetic pathways is of high interest in organic chemistry. Here, we describe an operationally simple and short synthesis of the privileged scaffold dihydropyrrolo[1,2-a]pyrazine-dione from readily accessible starting materials. The alkaloid-type polycyclic scaffold with potential bioactivity was achieved by a multicomponent reaction (MCR)-based protocol via a Ugi four-component reaction and Pictet-Spengler sequence under different conditions, yielding a diverse library of products.

Access to Isoquinolin-2(1H)-yl-acetamides and Isoindolin-2-yl-acetamides from a Common MCR Precursor.

We achieved a divergent synthesis of isoquinolin-2(1H)-yl-acetamides (16 examples, up to 90% yields) and regioselective isoindolin-2-yl-acetamides (14 examples, up to 93% yields) in moderate to good yields by reacting various substituted ethanones or terminal alkynes with Ugi-4CR intermediates via an ammonia-Ugi-4CR/Copper(I)-catalyzed annulation sequence reaction. The same intermediate thus gives 2D distant but 3D closely related scaffolds, which can be of high interest in exploiting chemistry space on a receptor. The scopes and limitations of these efficient sequence reactions are described, as well as gram-scale synthesis.

Low-Coordinate Erbium(III) Single-Molecule Magnets with Photochromic Behavior.

The structures and magnetic properties of photoresponsive magnets can be controlled or fine-tuned by visible light irradiation, which makes them appealing as candidates for ternary memory devices: photochromic and photomagnetic at the same time. One of the strategies for photoresponsive magnetic systems is the use of photochromic/photoswitchable molecules coordinated to paramagnetic metal centers to indirectly influence their magnetic properties. Herein, we present two erbium(III)-based coordination systems: a trinuclear molecule {[ErIII(BHT)3]3(dtepy)2}.4C5H12 (1) and a 1D coordination chain {[ErIII(BHT)3(azopy)}n·2C5H12 (2), where the bridging photochromic ligands belong to the class of diarylethenes: 1,2-bis((2-methyl-5-pyridyl)thie-3-yl)perfluorocyclopentene (dtepy) and 4,4'-azopyridine (azopy), respectively (BHT = 2,6-di-tert-butyl-4-methylphenolate). Both compounds show slow dynamics of magnetization, typical for single-molecule magnets (SMMs) as revealed by alternating current (AC) magnetic susceptibility measurements. The trinuclear compound 1 also shows an immediate color change from yellow to dark blue in response to near-UV irradiation. Such behavior is typical for the photoisomerization of the open form of the ligand into its closed form. The color change can be reversed by exposing the closed form to visible light. The chain-like compound 2, on the other hand, does not show significant signs of the expected trans-cis photoisomerization of the azopyridine in response to UV irradiation and does not appear to show photoswitching behavior.

Distinct sequence and structural feature of trypanosoma malate dehydrogenase.

Glycosomal malate dehydrogenase from Trypanosoma cruzi (tcgMDH) catalyzes the oxidation/reduction of malate/oxaloacetate, a crucial step of the glycolytic process occurring in the glycosome of the human parasite. Inhibition of tcgMDH is considered a druggable trait for the development of trypanocidal drugs. Sequence comparison of MDHs from different organisms revealed a distinct insertion of a prolin rich 9-mer (62-KLPPVPRDP-70) in tcgMDH as compared to other eukaryotic MDHs. Crystal structure of tcgMDH is solved here at 2.6 Å resolution with Rwork/Rfree values of 0.206/0.216. The tcgMDH forms homo-dimer with the solvation free energy (ΔGo) gain of -9.77 kcal/mol. The dimeric form is also confirmed in solution by biochemical assays, chemical-crosslinking and dynamic light scattering. The inserted 9-mer adopts a structure of a solvent accessible loop in the vicinity of NAD+ binding site. The distinct sequence and structural feature of tcgMDH, revealed in the present report, provides an anchor point for the development of inhibitors specific for tcgMDH, possible trypanocidal agents of the future.

Structure-based design approach to rational site-directed mutagenesis of ß-lactoglobulin.

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of ß-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine ß-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using ß-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.

Copper-Catalyzed Modular Assembly of Polyheterocycles.

Easy operation, readily accessible starting materials, and short syntheses of the privileged scaffold indeno[1,2-c]isoquinolinone were achieved by an multicomponent reaction (MCR)-based protocol via an ammonia-Ugi-four component reaction (4CR)/copper-catalyzed annulation sequence. The optimization and scope and limitations of this short and general sequence are described. The methodology allows an efficient construction of a wide variety of indenoisoquinolinones in just two steps.

Glycoconjugates via phosphorus ylides.

A facile, high yielding access to rare chimeric compounds combining phosphorus ylides with complex glycosyl formamides is described. We determined x-ray structures gaining structural insight into this compounds class. In addition, data mining of similar compounds deposited within the Cambridge Structural Database was performed. These derivatives could be used either as synthetic intermediates via the ylide functionalization and glyco chemical biology synthons or improving the pharmacokinetic properties of a potential bioactive molecule, exploiting the glycosyl moiety.

Structure and Reactivity of Glycosyl Isocyanides.

3D structural information was obtained from mono-, di- and trisaccharide formamide and isocyanide derivatives by analysis of their X-ray crystal structure and NMR spectroscopy. The isocyanide anomeric effect was observed. Data mining of the Cambridge Structural Database (CSD) was performed and statistically confirmed our findings. Application of the glycoside isocyanides in the synthesis of novel glycoconjugates as drug-like scaffolds by MCR chemistry underscores the usefulness of the novel building blocks.

Crystal structure of thebaine 6-O-demethylase from the morphine biosynthesis pathway.

Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy), which belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODD) family, is a key enzyme in the morphine biosynthesis pathway. Initially, T6ODM was characterized as an enzyme catalyzing O-demethylation of thebaine to neopinone and oripavine to morphinone. However, the substrate range of T6ODM was recently expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate (T6ODM:SIN, PDB: 5O7Y). Both metal and 2OG binding sites display similarity to other proteins from the ODD family, but T6ODM is characterized by an exceptionally large substrate binding cavity, whose volume can partially explain the promiscuity of this enzyme. Moreover, the size of the cavity allows for binding of multiple molecules at once, posing a question about the substrate-driven specificity of the enzyme.

Two-Step Macrocycle Synthesis by Classical Ugi Reaction.

The direct nonpeptidic macrocycle synthesis of α-isocyano-ω-amines via the classical Ugi four-component reaction (U-4CR) is introduced. Herein an efficient and flexible two-step procedure to complex macrocycles is reported. In the first step, the reaction between unprotected diamines and isocyanocarboxylic acids gives high diversity of unprecedented building blocks in high yield. In the next step, the α-isocyano-ω-amines undergo a U-4CR with high diversity of aldehydes and carboxylic acids in a one-pot procedure. This synthetic approach is short and efficient and leads to a wide range of macrocycles with different ring sizes.

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC50's in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.

Cysteine Isocyanide in Multicomponent Reaction: Synthesis of Peptido-Mimetic 1,3-Azoles.

An alternative approach toward the simple and robust synthesis of highly substituted peptidic thiazole derivatives using Ugi-multicomponent reaction (U-MCR) is described. Thus, we introduced the enantiopure (R)-2-methyl-2-isocyano-3-(tritylthio)propanoate as a novel class of isocyanide in MCR. This bifunctional isocyanide was found to undergo mild cyclodehydration to afford thiazole containing peptidomimetics in a short synthetic sequence. Several examples of bis-heterocyclic rings were also synthesized through the proper choice of the aldehyde component in the U-4CR. The method opens a wide range of applications toward the synthesis of nonribosomal natural products and other bioactive compounds.

Two-Step Synthesis of Complex Artificial Macrocyclic Compounds.

The design and synthesis of head-to-tail linked artificial macrocycles using the Ugi-reaction has been developed. This synthetic approach of just two steps is unprecedented, short, efficient and works over a wide range of medium (8-11) and macrocyclic (≥12) loop sizes. The substrate scope and functional group tolerance is exceptional. Using this approach, we have synthesized 39 novel macrocycles by two or even one single synthetic operation. The properties of our macrocycles are discussed with respect to their potential to bind to biological targets that are not druggable by conventional, drug-like compounds. As an application of these artificial macrocycles we highlight potent p53-MDM2 antagonism.

α-Amino Acid-Isosteric α-Amino Tetrazoles.

The synthesis of all 20 common natural proteinogenic and 4 otherα-amino acid-isosteric α-amino tetrazoles has been accomplished, whereby the carboxyl group is replaced by the isosteric 5-tetrazolyl group. The short process involves the use of the key Ugi tetrazole reaction followed by deprotection chemistries. The tetrazole group is bioisosteric to the carboxylic acid and is widely used in medicinal chemistry and drug design. Surprisingly, several of the common α-amino acid-isosteric α-amino tetrazoles are unknown up to now. Therefore a rapid synthetic access to this compound class and non-natural derivatives is of high interest to advance the field.

Artificial Macrocycles by Ugi Reaction and Passerini Ring Closure.

Artificial macrocycles can be convergently synthesized by a sequence of an Ugi multicomponent reaction (MCR) followed by an intramolecular Passerini MCR used to close the macrocycle. Significantly, in this work, the first intramolecular macrocyclization through a Passerini reaction is described. We describe 21 macrocycles of a size of 15-20. The resulting macrocyclic depsipeptides are model compounds for natural products and could find applications in drug discovery.

Investigating the effects of double mutation C30A/C75A on onconase structure: Studies at atomic resolution.

The structure of onconase C30A/C75A double mutant has been determined at 1.12Å resolution. The structure has high structural homology to other onconase structures. The changes being results of mutation are relatively small, distributed asymmetrically around the two mutated positions, and they are observed not only in the mutation region but expanded to entire molecule. Different conformation of Lys31 side chain that influences the hydrogen bonding network around catalytic triad is probably responsible for lower catalytic efficiency of double mutant. The decrease in thermal stability observed for the onconase variant might be explained by a less dense packing as manifested by the increase of the molecular volume and the solvent accessible surface area.

Exploring the coordination chemistry of ruthenium complexes with lysozymes: structural and in-solution studies.

This study presents a comprehensive structural analysis of the adducts formed upon the reaction of two Ru(III) complexes [HIsq][trans-RuIIICl4(dmso)(Isq)] (1) and [H2Ind][trans-RuIIICl4(dmso)(HInd)] (2) (where HInd-indazole, Isq-isoquinoline, analogs of NAMI-A) and two Ru(II) complexes, cis-[RuCl2(dmso)4] (c) and trans-[RuCl2(dmso)4] (t), with hen-egg white lysozyme (HEWL). Additionally, the crystal structure of an adduct of human lysozyme (HL) with ruthenium complex, [H2Ind][trans-RuCl4(dmso)(HInd)] was solved. X-ray crystallographic data analysis revealed that all studied Ru complexes, regardless of coordination surroundings and metal center charge, coordinate to the same amino acids (His15, Arg14, and Asp101) of HEWL, losing most of their original ligands. In the case of the 2-HL adduct, two distinct metalation sites: (i) Arg107, Arg113 and (ii) Gln127, Gln129, were identified. Crystallographic data were supported by studies of the interaction of 1 and 2 with HEWL in an aqueous solution. Hydrolytic stability studies revealed that both complexes 1 and 2 liberate the N-heterocyclic ligand under crystallization-like conditions (pH 4.5) as well as under physiological pH conditions, and this process is not significantly affected by the presence of HEWL. A comparative examination of nine crystal structures of Ru complexes with lysozyme, obtained through soaking and co-crystallization experiments, together with in-solution studies of the interaction between 1 and 2 with HEWL, indicates that the hydrolytic release of the N-heterocyclic ligand is one of the critical factors in the interaction between Ru complexes and lysozyme. This understanding is crucial in shedding light on the tendency of Ru complexes to target diverse metalation sites during the formation and in the final forms of the adducts with proteins.