Structural and thermodynamic characterization of inhibitor binding to aldose reductase: Insights into binding modes, driving forces and selectivity determinants

The TIM-barrel folded enzyme Aldose reductase (ALR2) is a valuable model system to study structural and thermodynamic features of inhibitor binding and, furthermore, represents an excellent drug target. To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Its attraction as a test system consists furthermore in the high mobility and adaptivity properties of its active site residues, giving rise to various distinct binding pocket conformers and pronounced induced-fit adaptations upon ligand binding. In chapter 2, we combine a structural characterization of the experimental binding modes observed for two virtual screening hits with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding. The nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a strongly favourable contribution to binding enthalpy in case the inhibitor is equipped with a nitro group at the corresponding position. As these data suggest, the nitro group provokes the enthalpic contribution, in addition to the H-bond mentioned above, by accepting two “non-classical” H-bonds donated by the aromatic tyrosine side chain. In chapter 3, we report on the crystal structures of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase. The inhibitor occupies with its pyridazinone head group the catalytic site whereas the chloro-benzofurane moiety penetrates into the opened specificity pocket. The high resolution structure provides some evidence that the pyridazinone group binds in a negatively charged deprotonated state whereas the neighboring His 110 residue most likely adopts a neutral uncharged state. In chapter 4, we probed the ALR2 binding site with a novel structural class of inhibitors in order to identify putative pocket adaptations. We elucidated two ALR2 crystal structures, each complexed with a member of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat, both inhibitors leave the specificity pocket in closed state. Unexpectedly, the more potent ligand extends the catalytic pocket by opening of a novel subpocket. The second studied inhibitor differs from the first only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, its binding mode differs dramatically from that of the first inhibitor. The two ligand complexes represent an impressive example, how the slight change of a chemically extended side chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships. In chapter 5, we study the binding process of inhibitors to ALR2 with respect to changes of the protonation inventory upon complex formation. As the protonation event will strongly contribute to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and -releasing functional groups of the system is of utmost importance. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr 48) as likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to remarkable extent if only the cofactor NADP+ is bound. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects. In chapter 6, the ALR2 binding site is probed for selectivity determining features, which make binding of certain ligands to ALR2 more attractive than to the concurrent isoform aldehyde reductase (ALR1). The resulting mutational constructs of ALR2 are probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and ITC. Accurate crystal structure-determination of protein-ligand complexes is the starting point for further design hypotheses to predict novel leads with improved properties. This widely accepted practise relies on the assumption that the crystal structure of a given protein-ligand complex is unique and independent of the protocol applied to produce the crystals. In chapter 7, we present two examples indicating that this assumption is not generally given

Merging the binding sites of aldose and aldehyde reductase for detection of inhibitor selectivity-determining features.

International audienceInhibition of human aldose reductase (ALR2) evolved as a promising therapeutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein-ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermodynamics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding

Non-ATP-Mimetic Organometallic Protein Kinase Inhibitor

US National Institutes of Health [CA114046]; German Research Foundation [ME 1805/9-1]; Helmholtz Zentrum Berlin (HZB

Structural Basis for HTLV‑1 Protease Inhibition by the HIV‑1 Protease Inhibitor Indinavir

HTLV-1 protease (HTLV-1 PR) is an aspartic protease which represents a promising drug target for the discovery of novel anti-HTLV-1 drugs. The X-ray structure of HTLV-1 PR in complex with the well-known and approved HIV-1 PR inhibitor Indinavir was determined at 2.40 Å resolution. In this contribution, we describe the first crystal structure in complex with a nonpeptidic inhibitor that accounts for rationalizing the rather moderate affinity of Indinavir against HTLV-1 PR and provides the basis for further structure-guided optimization strategies

Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis1[W][OA]

Flavanone 3β-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the β-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction)

An Organometallic Inhibitor for the Human Repair Enzyme 7,8-Dihydro-8-oxoguanosine Triphosphatase

German Research Foundation [ME 1805/9-1]; U.S. National Institutes of Health [CA114046]The probe-based discovery of the first small-molecule inhibitor of the repair enzyme 8-oxo-dGTPase (MTH1) is presented, which is an unconventional cyclometalated ruthenium half-sandwich complex. The organometallic inhibitor with low-nanomolar activity displays astonishing specificity, as verified in tests with an extended panel of protein kinases and other ATP binding proteins. The binding of the organometallic inhibitor to MTH1 is investigated by protein crystallography

Privileged Structures Meet Human T‑Cell Leukemia Virus‑1 (HTLV-1): C₂‑Symmetric 3,4-Disubstituted Pyrrolidines as Nonpeptidic HTLV‑1 Protease Inhibitors

3,4-disubstituted pyrrolidines originally designed to inhibit the closely related HIV-1 protease were evaluated as privileged structures against HTLV-1 protease (HTLV-1 PR). The most potent inhibitor of this series exhibits two-digit nanomolar affinity and represents, to the best of our knowledge, the most potent nonpeptidic inhibitor of HTLV-1 PR described so far. The X-ray structures of two representatives bound to HTLV-1 PR were determined, and the structural basis of their affinity is discussed

Structural hot spots determine functional diversity of the Candida glabrata epithelial adhesin family

For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species

Interplay of Template Constraints and Microphase Separation in Polymeric Nano-Objects Replicated from Novel Modulated and Interconnected Nanoporous Anodic Alumina

We report on a versatile strategy to fabricate shape-controlled polymeric nano-objects with an internal compartmentalized structure by replication from pore-diameter-modulated and interconnected nanoporous anodized aluminum oxide (AAO). The AAO with modulated pore diameters is synthesized in a refined temperature-modulated hard anodization (TMHA) approach, by alternating the flow rate of an air stream cooling the electrolyte. Through pore-widening of the templates with modulated pore diameters, a stable interconnected 3D network with transversal ellipsoidal holes is fabricated as a second template structure. From these unprecedented template structures exhibiting modulated pores and an intricate 3D network structure, shape-controlled polymeric nano-objects are replicated using the melt wetting method with polystyrene homopolymer (PS) and cylinder-forming block copolymer polystyrene-<i>block</i>-polydimethylsiloxane (PS₂₉₈-<i>b</i>-PDMS₁₉₅). The replicated nanostructures are separated into individual anisotropically shaped nanocapsules by passing the replicas through a polycarbonate membrane followed by gentle sonication. Inside the nano-objects, the microphase separation of PS₂₉₈-<i>b</i>-PDMS₁₉₅ affords parallel aligned nanophases, in which both domain sizes and morphologies are different compared to the unconfined bulk according to transmission electron microscopy analyses. These advanced AAO platforms and the nanoscale polymer replicas offer new routes for compartmentalized nano-objects with potential future relevance for applications ranging from drug nanocarriers to biosensors