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

Inhibition 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.     

Binding Thermodynamics of Ferredoxin:NADP Reductase: Two Different Protein Substrates and One Energetics

The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP⁺ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His²⁹⁹ FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics.     

Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution

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. In order to identify novel lead compounds, a virtual screening (VS) was performed successfully suggesting carboxylate-type inhibitors of sub-micromolar to micromolar affinity. Here, we combine a structural characterization of the binding modes observed by X-ray crystallography with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding, particularly of the first leads from VS. Characteristic features of this novel inhibitor type include a carboxylate head group connected via an alkyl spacer to a heteroaromatic moiety, which is linked to a further nitro-substituted aromatic portion. The crystal structures of two enzyme-inhibitor complexes have been determined at resolutions of 1.43 A and 1.55 A. Surprisingly, the carboxylic group of the most potent VS lead occupies the catalytic pocket differently compared to the interaction geometry observed in almost all other crystal structures with structurally related ligands and obtained under similar conditions, as an interstitial water molecule is picked up upon ligand binding. The nitro-aromatic moiety of both leads occupies the specificity pocket of the enzyme, however, adopting a different geometry compared to the docking prediction: unexpectedly, the nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. A peptide group located at the active site orients in such a way that H-bond formation to one nitro group oxygen atom is enabled, whereas a neighbouring tyrosine side-chain performs a slight rotation off from the binding cavity to accommodate the nitro group. 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. To further investigate this phenomenon, we determined crystal structures and thermodynamic data of two similarly constituted IDD-type inhibitors addressing the specificity pocket with either a nitro or halogen-substituted aromatic moiety. 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 summary, this study provides the platform for further structure-guided design hypotheses of novel drug candidates with higher affinity and selectivity.     

High-resolution crystal structure of aldose reductase complexed with the novel sulfonyl-pyridazinone inhibitor exhibiting an alternative active site anchoring group

The crystal structure of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase, the first enzyme of the polyol pathway, has been determined to 1.43 angstroms and 0.95 angstroms resolution. The ternary complex of inhibitor, cofactor and enzyme has been obtained by soaking of preformed crystals. Supposedly due to low solubility in the crystallisation buffer, in both structures the inhibitor shows reduced occupancy of 74% and 46% population, respectively. The pyridazinone head group of the inhibitor occupies the catalytic site, whereas the chloro-benzofuran 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 neighbouring His110 residue most likely adopts a neutral uncharged status. Since the latter structure is populated by the ligand to only 46%, a second conformation of the C-terminal ligand-binding region can be detected. This conformation corresponds to the closed state of the specificity pocket when no or only small ligands are bound to aldose reductase. The two conformational states are in good agreement with frames observed along a molecular dynamics trajectory describing the transition from closed to open situation. Accordingly, both geometries, superimposed in the averaged crystal structure, correspond to snapshots of the ligand-bound and the unbound state. Isothermal titration calorimetry has been applied to determine the binding constants of the investigated pyridazinone in comparison to the hydantoin sorbinil and the carboxylate-type inhibitors IDD 594 and tolrestat. The pyridazinone exhibits a binding affinity similar to those of tolrestat and sorbinil, and shows slightly reduced affinity compared to IDD 594. These studies elucidating the binding mode and providing information about protonation states of protein side-chains involved in binding of this novel class of inhibitors establish the platform for further structure-based drug design.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.     

Calorimetric studies of ligand binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.     

Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pK(a) calculations and ITC experiments

The protonation states of a protein and a ligand can be altered upon complex formation. Such changes can be detected experimentally by isothermal titration calorimetry (ITC). For a series of ligands binding to the serine proteases trypsin and thrombin, we previously performed an extensive ITC and crystallographic study and were able to identify protonation changes for four complexes. However, since ITC measures only the overall proton exchange, it does not provide structural insights into the functional groups involved in the proton transfer. Using Poisson-Boltzmann calculations based on our recently developed PEOE_PB charges, we compute pK(a) values for all complexes of our former study in order to reveal the residues with altered protonation states. The results indicate that His57, a member of the catalytic triad, is responsible for the most relevant pK(a) shifts leading to the experimentally detected protonation changes. This finding is in contrast to our previous assumption that the observed protonation changes occur at the carboxylic group of the ligands. The newly detected proton acceptor is used for a revised factorization of the ITC data, which is necessary whenever the protonation inventory changes upon complexation. The pK(a) values of complexes showing no protonation change in the ITC experiment are reliably predicted in most cases, whereas predictions of strongly coupled systems remain problematic.     

Structures of Leishmania major pteridine reductase complexes reveal the active site features important for ligand binding and to guide inhibitor design

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Thermodynamics of aryl-dihydroxyphenyl-thiadiazole binding to human Hsp90

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic K(d) approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors.     

Evidence for a novel binding site conformer of aldose reductase in ligand-bound state

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor 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 (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of 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.     

Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans

Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K_a) were in the range of 5.1–49 ? 10⁴ M^− 1 and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (∼ − 1 kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.     

Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246-252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.     

Probing flexibility and "induced-fit" phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations

Aldose reductase is a promising target for the treatment of diabetic complications, and as such, has become the focus of various drug design projects. As revealed by a survey of available crystal structures, the protein shows pronounced induced-fit effects upon ligand binding. Although helping to explain the enzyme's substrate promiscuity, phenomena of this kind are still responsible for significant complications in structure-based design efforts directed to aldose reductase. Accordingly, a deeper understanding of the principles governing conformational alterations in this enzyme would be of utmost practical importance. As a first step in addressing this issue, molecular dynamics (MD) simulations have been carried out. The ultrahigh resolution crystal structure of aldose reductase complexed with inhibitor IDD594 served as ideal starting point for a set of different simulations of nanosecond time scale: the native complexed state with bound inhibitor, the uncomplexed state (after removal of the inhibitor) at standard temperature, and the uncomplexed state at elevated temperature. The reference simulation of the complex exhibits extraordinary stability of the overall fold, whereas two distinct conformational substates are found for the binding-site region. In contrast, already at standard temperature pronounced changes are observed in the binding region during the simulation of the uncomplexed state. Leu300, for example, closes the access to the pocket opened by IDD594. On the other hand, conformations around the catalytic site are highly conserved, with the His110-Tyr48-NADP+ orientation being stabilized by a water molecule. Detailed analysis of the trajectories allows to reveal a set of distinct conformational substates that may prove useful as alternative structural templates in virtual screening for new aldose reductase inhibitors.     

Expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying soaking and co-crystallisation conditions

In structure-based drug design, accurate crystal structure determination of protein-ligand complexes is of utmost importance in order to elucidate the binding characteristics of a putative lead to a given target. It is the starting point for further design hypotheses to predict novel leads with improved properties. Often, crystal structure determination is regarded as ultimate proof for ligand binding providing detailed insight into the specific binding mode of the ligand to the protein. 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. We present two examples indicating that this assumption is not generally given, even though the composition of the mother liquid for crystallisation was kept unchanged: Multiple crystal structure determinations of aldose reductase complexes obtained under varying crystallisation protocols concerning soaking and crystallisation exposure times were performed resulting in a total of 17 complete data sets and ten refined crystal structures, eight in complex with zopolrestat and two complexed with tolrestat. In the first example, a flip of a peptide bond is observed, obviously depending on the crystallisation protocol with respect to soaking and co-crystallisation conditions. This peptide flip is accompanied by a rupture of an H-bond formed to the bound ligand zopolrestat. The indicated enhanced local mobility of the complex is in agreement with the results of molecular dynamics simulations. As a second example, the aldose reductase-tolrestat complex is studied. Unexpectedly, two structures could be obtained: one with one, and a second with four inhibitor molecules bound to the protein. They are located in and near the binding pocket facilitated by crystal packing effects. Accommodation of the four ligand molecules is accompanied by pronounced shifts concerning two helices interacting with the additional ligands.     

Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (− TΔΔS = 5.1 ± 0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH = − 3.5 ± 0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.     

Structure based design of compounds from natural sources for diabetes and inflammation

Medicinal plants and marine sources are important elements of indigenous medical systems worldwide. The natural drugs from medicinal plants and marine sources have received considerable interest in treatment of diabetes and inflammation. Based on literature, alpha glucosidase, aldose reductase and PTP1B enzymes were chosen as anti-diabetes targets and PLA₂ was chosen for the anti-inflammatory target. In our study, plant and bromophenols (BPs) inhibitors were screened using High Throughput Virtual screening (HTVS) followed by Induced Fit Docking (IFD) studies were carried out against diabetes and inflammation targets. The IFD result of natural inhibitors has showed favorable docking score, glide energy and hydrogen bonds interactions with the active site residues. Some of the natural inhibitors successively satisfied all the in silico parameters among the others and seem to be potent inhibitors against diabetes and inflammation.     

Exploring antidiabetic potential of adamantyl-thiosemicarbazones via aldose reductase (ALR2) inhibition

The role of aldose reductase (ALR2) in diabetes mellitus is well-established. Our interest in finding ALR2 inhibitors led us to explore the inhibitory potential of new thiosemicarbazones. In this study, we have synthesized adamantyl-thiosemicarbazones and screened them as aldehyde reductase (ALR1) and aldose reductase (ALR2) inhibitors. The compounds bearing phenyl 3a, 2-methylphenyl 3g and 2,6-dimethylphenyl 3m have been identified as most potent ALR2 inhibitors with IC₅₀ values of 3.99 ± 0.38, 3.55 ± 0.26 and 1.37 ± 0.92 µM, respectively, compared with sorbinil (IC₅₀ = 3.14 ± 0.02 μM). The compounds 3a, 3g, and 3m also inhibit ALR1 with IC₅₀ value of 7.75 ± 0.28, 7.26 ± 0.39 and 7.04 ± 2.23 µM, respectively. Molecular docking was also performed for putative binding of potent inhibitors with target enzyme ALR2. The most potent 2,6-dimethylphenyl bearing thiosemicarbazone 3m (IC₅₀ = 1.37 ± 0.92 µM for ALR2) and other two compound 3a and 3g could potentially lead for the development of new therapeutic agents.     

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3A, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.     

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.