Tuomo Laitinen - Academia.edu (original) (raw)

Papers by Tuomo Laitinen

Protein Engineering Design & Selection, Apr 1, 2000

X-Ray crystal structures have revealed that 2,3-epoxypropyl-β-D-xyloside reacts with endo-1,4-β-x... more X-Ray crystal structures have revealed that 2,3-epoxypropyl-β-D-xyloside reacts with endo-1,4-β-xylanase (XYNII) by forming a covalent bond with Glu86. In contrast, 3,4-epoxybutyl-β-D-xyloside forms a covalent bond with Glu177. In the normal enzyme reaction Glu86 acts as the catalytic nucleophile and Glu177 as the acid/base catalyst. To rationalize the observed reactivity of the two mechanismbased inhibitors, we carried out eight 300 ps molecular dynamics simulations for different enzyme-inhibitor complexes. Simulations were done for both stereo isomers (R and S) of the inhibitors and for enzyme in which the protonation state of the nucleophile and acid/base catalyst was normal (Glu86 charged, Glu177 neutral) and in which the roles of the catalytic residues were reversed (Glu86 neutral, Glu177 charged). The number of reactive conformations found in each simulation was used to predict the reactivity of epoxy inhibitors. The conformation was considered to be a reactive one when at the same time (i) the proton of the catalytic acid was close (<2.9/3.4/3.9 Å) to the oxirane oxygen of the inhibitor, (ii) the nucleophile was close to the terminal carbon of the oxirane group (<3.4/3.9/4.4 Å) and (iii) the nucleophile approached the terminal carbon from a reactive angle (<30/45/60°from an ideal attack angle). On the basis of the number of reactive conformations, 2,3-epoxypropyl-β-D-xyloside was predicted to form a covalent bond with Glu86 and 3,4-epoxybutylβ-D-xyloside with Glu177, both in agreement with the experiment. Thus, the MD simulations and the X-ray structures indicate that in the covalent binding of 3,4epoxybutyl-β-D-xyloside the roles of the catalytic glutamates of XYNII are reversed from that of the normal enzyme reaction.

Journal of Organic Chemistry, Oct 27, 1998

A model system consisting of methyloxirane, formate, and formic acid was used to study the nucleo... more A model system consisting of methyloxirane, formate, and formic acid was used to study the nucleophile-catalyzed and nucleophile and acid-catalyzed opening of an epoxide ring using ab initio quantum mechanical (up to the MP4(SDQ)/6-31+G**//MP2/6-31+G** level) and density functional theory calculations (Becke3LYP/6-31+G**). This system serves as a model for the covalent binding of the epoxide inhibitor to the active site of glycosidase. The effects of solvation on reaction energies were estimated using the isodensity surface polarized continuum model. The opening of the oxirane ring was calculated to preferably take place between the epoxide oxygen and the less-substituted carbon. In agreement with the earlier experimental inferences, the results indicate that both the nucleophile and the acid/base catalyst are needed for the ring opening reaction to take place efficiently. The implications of the results for the enzyme-catalyzed opening of the epoxide ring were discussed.

Organic and Biomolecular Chemistry, 2003

The binding of xylotetraose in different conformations to the active site of endo-1,4-beta-xylana... more The binding of xylotetraose in different conformations to the active site of endo-1,4-beta-xylanase II (XynII) from Trichoderma reesei was studied using molecular dynamics (MD) simulations and free energy analyses employing the MM-PBSA (Molecular Mechanics-Poisson-Boltzmann Surface Area) method. MD simulations of 1 ns were done for the substrate xylotetraose having the reactive sugar, which is bound in the -1 subsite of XynII in the 4C1 (chair) and 2So (skew boat) ground state conformations, and for the transition state of the XynII catalysed hydrolysis of the beta-glycosidic linkage. According to the simulations and free energy analysis, XynII binds the substrate with the -1 sugar in the 2So conformation 59.8 kJ mol(-1) tighter than the substrate with the sugar in the 4C1 conformation. The reactive 2So conformation resembles closely the reaction transition state and has the breaking glycosidic bond in a pseudo-axial orientation ready for facile bond cleavage. The transition state was calculated to be bound 77.1 kJ mol(-1) tighter than the 4C1 ground state conformation. The molecular mechanical interaction energy between the enzyme and the reactive pyranoside unit at the -1 subsite was 75.7 kJ mol(-1) more favorable for the binding of the 2So conformation than the 2C1 conformation, explaining the clearly tighter binding of the reactive structure The results of this study indicate that in the Michaelis complex XynII, a member of the family 11 xylanase, the substrate is bound in a skew boat conformation and in the catalytic reaction, the -1 sugar proceeds from the 4C1 conformation through 2So to the transition state with the -1 sugar in the 2,5B conformation.

The 4-anilino-quinoline and 4-anilino-quinazoline ring systems have been the focus of significant... more The 4-anilino-quinoline and 4-anilino-quinazoline ring systems have been the focus of significant efforts in prior kinase drug discovery programs, which have led to approved medicines. Broad kinome profiles of these compounds have now been assessed with the advent of advanced screening technologies. These ring systems while, originally designed for specific targets including epidermal growth factor receptor (EGFR), actually display a number of potent collateral kinase targets, some of which have been associated with negative clinical outcomes. We have designed and synthesized a series of 4-anilino-quin(az)olines in order to better understand the structure activity relationships of three main collateral kinase targets of quin(az)oline-based kinase inhibitors: cyclin G associated kinase (GAK), STE20-like serine/threonine-protein kinase (SLK) and Serine/threonine-protein kinase 10 (STK10) through a series of quantitative structure activity relationship (QSAR) analysis and water mapping...

ABSTRACTEpidermal growth factor receptor (EGFR) inhibitors have been used to target non-small cel... more ABSTRACTEpidermal growth factor receptor (EGFR) inhibitors have been used to target non-small cell lung cancer (NSCLC) and chordomas with varying amounts of success. We have probed several key structural features including an interaction with Asp855 within the EGFR DGF motif and interactions with the active site water network. The EGFR target engagement was then evaluated in an in-cell assay. Additionally, inhibitors were profiled in representative cellular models of NSCLC and chordomas. In addition to a structure activity relationship insights for EGFR inhibtior design, we also identified a compound (18) that is the most potent inhibitor (IC50 = 310 nM) on the UCH-2 chordoma cell line to date.

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2017

In recent years, the demand and interest for functionalized polymers have increased for drug deli... more In recent years, the demand and interest for functionalized polymers have increased for drug delivery purposes. Because of the increased interest, methods that can be used to predict physical and chemical properties of polymers prior to synthesis would be of high value for the design and development of novel polymer structures. Through use of molecular descriptors and Principal Component Analysis, this study explores the possibilities of using in silico methods for polymer design and characterization for property prediction. The results presented in this paper suggest that it is possible to produce a model, which can successfully distinguish between a set of both structurally similar and different polymers based on their surface properties.

ChemMedChem, Jan 11, 2016

A diverse library of 5-thieno-, 5-oxo-, and 5-imino-1,2,3-dithiazole derivatives was synthesized ... more A diverse library of 5-thieno-, 5-oxo-, and 5-imino-1,2,3-dithiazole derivatives was synthesized and evaluated for efficacy against the feline immunodeficiency virus (FIV) as a model for HIV in cells. Several diverse compounds from this series displayed nanomolar activity and low toxicity, representing a potential new class of compounds for the treatment of FIV and HIV.

Drug metabolism and disposition: the biological fate of chemicals, Jan 23, 2015

The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commo... more The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commonly used drugs. Relatively little is known about CYP2C19 inhibitors, including compounds of natural origin which could inhibit CYP2C19 potentially causing clinically relevant metabolism-based drug interactions. We evaluated a series (N=49) of structurally related plant isoquinoline alkaloids for their abilities to interact with CYP2C19 enzyme using in vitro and in silico methods. We examined several common active alkaloids found in herbal products such as apomorphine, berberine, noscapine, and papaverine as well as the previously identified mechanism-based inactivators bulbocapnine, canadine, and protopine. The IC50 values of the alkaloids ranged from 0.11 to 210 μM, and 42 of the alkaloids were confirmed to be time-dependent inhibitors of CYP2C19. Molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis revealed key interactions of the pote...

Molecular pharmacology, 2014

The primary route of inactivation of the endocannabinoid 2-arachidonoylglycerol in the central ne... more The primary route of inactivation of the endocannabinoid 2-arachidonoylglycerol in the central nervous system is through enzymatic hydrolysis, mainly carried out by monoacylglycerol lipase (MAGL), along with a small contribution by the α/β-hydrolase domain (ABHD) proteins ABHD6 and ABHD12. Recent methodological progress allowing kinetic monitoring of glycerol liberation has facilitated substrate profiling of the human endocannabinoid hydrolases, and these studies have revealed that the three enzymes have distinct monoacylglycerol substrate and isomer preferences. Here, we have extended this substrate profiling to cover four prostaglandin glycerol esters, namely, 15-deoxy-Δ(12,14)-prostaglandin J2-2-glycerol (15d-PGJ2-G), PGD2-G, PGE2-G, and PGF2 α-G. We found that the three enzymes hydrolyzed the tested substrates, albeit with distinct rates and preferences. Although human ABHD12 (hABHD12) showed only marginal activity toward PGE2-G, hABHD6 preferentially hydrolyzed PGD2-G, and huma...

Letters in Drug Design & Discovery, 2013

Bioorganic & Medicinal Chemistry, 2014

The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and m... more The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

Bioorganic & Medicinal Chemistry Letters, 2015

Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAG... more Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC 50 = 46 nM), and was found to have almost 150-fold higher selectivity over human recombi

Proteins: Structure, Function, and Bioinformatics, 2004

Antiestradiol antibody 57-2 binds 17␤-estradiol (E2) with moderately high affinity (K a ‫؍‬ 5 ؋ 1... more Antiestradiol antibody 57-2 binds 17␤-estradiol (E2) with moderately high affinity (K a ‫؍‬ 5 ؋ 10 8 M ؊1). The structurally related natural estrogens estrone and estriol as well synthetic 17deoxy-estradiol and 17␣-estradiol are bound to the antibody with 3.7-4.9 kcal mol ؊1 lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were applied to investigate the factors responsible for the relatively low cross-reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D-ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM-PBSA method. Both the FEP and MM-PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid-antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol ؊1 to the interaction energy. Because the relative binding free energies of estrone (two antibody-steroid hydrogen bonds), estriol (three hydrogen bonds), 17-deoxyestradiol (no hydrogen bonds), and 17␣-estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water-steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM-PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57-2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3-C 4 F 5 , studied earlier with the MM-PBSA method. Proteins 2004;55:34-43.

Protein Engineering, Design and Selection, 2000

Protein Engineering Design and Selection, 2007

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, w... more Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the a-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.

Phys. Chem. Chem. Phys., 2004

ABSTRACT Binding of xylotetraose to the family 10 xylanase (CEX) from Cellulomonas fimi and the f... more ABSTRACT Binding of xylotetraose to the family 10 xylanase (CEX) from Cellulomonas fimi and the family 11 xylanase (BCX) from Bacillus circulans was investigated using quantum mechanical calculations, molecular dynamics simulations and molecular mechanics–Poisson–Boltzmann surface area (MM-PBSA) free energy calculations. In the reactive enzyme–substrate conformation, in which the catalytic groups are precisely positioned for the catalytic reaction, the −1 sugar moiety of the substrate adopted a B3,O (boat) conformation for CEX and a 2SO (skew-boat) conformation for BCX. Upon binding the internal energies of the reactive substrate conformations are increased by about 9 kJ mol−1. In contrast to this, the internal energies of substrates in all-chair conformation are increased by 33–43 kJ mol−1, with somewhat larger increase for BCX than CEX. As a consequence, the stability order of the all-chair conformation, which is the most stable conformation in aqueous solution, and the distorted reactive conformations is reversed upon binding to the enzyme. Thus, in addition to specific enzyme–substrate interactions, CEX and especially BCX recognise the reactive high-energy conformation by the substrate-binding site of several sugar-binding subsites complementary in shape to the reactive conformation. The role of substrate distortion in glycosidase-catalysed reactions is discussed.

Molecular Pharmacology, 2013

Considerable progress has been made in recent years in developing selective and potent monoacylgl... more Considerable progress has been made in recent years in developing selective and potent monoacylglycerol lipase (MAGL) inhibitors. In pursuance of investigating measures to inhibit this enzyme, less attention has been paid to improve our understanding on its catalytic mechanisms or substrate preference. In this study, we utilized site-directed mutagenesis and show by using versatile activity assays combined with molecular modeling that Cys242 and Tyr194, the two opposing amino acid residues in the catalytic cavity of MAGL, play important roles in determining the rate and isomer preference of monoacylglycerol hydrolysis. In contrast to wild type enzyme, which hydrolyzes 1-and 2-monoacylglycerols with similar rates, mutation of Cys242 to alanine caused a significant reduction in overall activity (maximal velocity, V max) and particularly, skewed the balanced hydrolysis of isomers to favor the 2-isomer. Molecular modeling studies indicated that this was caused by unfavorable structural features towards 1-isomers as well as impaired recognition of OHgroups in the glycerol moiety. Direct functional involvement of Cys242 in the catalysis was found unlikely due to remote distance from the catalytic serine. Unlike C242A, mutation of Tyr194 did not bias the hydrolysis of 1-and 2-monoacylglycerols but significantly compromised overall activity. Finally, mutation of Cys242 was also found to impair inhibition of MAGL, especially that by fluorophosphonate derivatives (13-63-fold reduction in potency). Taken together, this study provides new experimental and modeling insights into the molecular mechanisms of MAGL-catalyzed hydrolysis of the primary endocannabinoid 2arachidonoylglycerol and related monoacylglycerols.

Molecular Pharmaceutics, 2011

The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobioti... more The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobiotic and endobiotic metabolism. The unique properties of human CAR, such as the high constitutive activity and the complexity of signaling, as well as the lack of functional and predictive cellbased assays to study the properties of the receptor, have hindered the discovery of selective human CAR ligands. Here we report a novel human CAR inverse agonist, 1-[(2methylbenzofuran-3-yl)methyl]-3-(thiophen-2-ylmethyl) urea (S07662), which suppresses human CAR activity, recruits the corepressor NCoR in cell-based assays, and attenuates the phenytoin-and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO)-induced expression of CYP2B6 mRNA in human primary hepatocytes. The properties of S07662 are also compared with those of known human CAR inverse agonists by using an array of different in vitro and in silico assays. The identified compound S07662 can be used as a chemical tool to study the biological functions of human CAR and also as a starting point for the development of new drugs for various conditions involving the receptor.

The Journal of Organic Chemistry, 1998

Journal of Medicinal Chemistry, 2013

Synthesis and spectroscopic characterisation of compounds 7, 9, 14, 22-40 S3-S11 Separation of en... more Synthesis and spectroscopic characterisation of compounds 7, 9, 14, 22-40 S3-S11 Separation of enantiomers of compounds 41, 42 and 50 S11-S14 Elemental Analyses S15 The crystallographic details of compound 53 S16-S17 Determination of ABHD6/12 activity using a sensitive fluorescent glycerol assay S17-S18 Assay of anandamide uptake S19 Cannabinoid receptor activity S20 Molecular modelling studies S20-S22 References S23-S24