Last publication: Bioinformatics, 2019, 1–3 doi: 10.1093/bioinformatics/btz946


Our Team Members Publications




Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is a Challenging Target for Small-Molecule Inhibitors Design.

Bzówka M., Mitusińska K., Raczyńska A., Samol A., Tuszyński J.A., Góra A. ARTICLEOpen Access



The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

Simple Selection Procedure to Distinguish between Static and Flexible Loops.

Mitusińska K., Skalski T., Góra A., Int. J. Mol. Sci. 2020, 21(7), 2293;ARTICLEOpen Access


Loops are the most variable and unorganized elements of the secondary structure of proteins. Their ability to shift their shape can play a role in the binding of small ligands, enzymatic catalysis, or protein–protein interactions. Due to the loop flexibility, the positions of their residues in solved structures show the largest B-factors, or in a worst-case scenario can be unknown. Based on the loops’ movements’ timeline, they can be divided into slow (static) and fast (flexible). Although most of the loops that are missing in experimental structures belong to the flexible loops group, the computational tools for loop reconstruction use a set of static loop conformations to predict the missing part of the structure and evaluate the model. We believe that these two loop types can adopt different conformations and that using scoring functions appropriate for static loops is not sufficient for flexible loops. We showed that common model evaluation methods, are insufficient in the case of flexible solvent-exposed loops. Instead, we recommend using the potential energy to evaluate such loop models. We provide a novel model selection method based on a set of geometrical parameters to distinguish between flexible and static loops without the use of molecular dynamics simulations. We have also pointed out the importance of water network and interactions with the solvent for the flexible loop modelling.

Applications of water molecules for analysis of macromolecule properties.

Mitusińska K., Raczyńska A., Bzówka M., Bagrowska W., Góra A., Computational and Structural Biotechnology Journal 2020, 18, 355-365; https://doi:10.1016/j.csbj.2020.02.001 ARTICLEOpen Access


Water molecules maintain proteins’ structures, functions, stabilities and dynamics. They can occupy certain positions or pass quickly via a protein’s interior. Regardless of their behaviour, water molecules can be used for the analysis of proteins’ structural features and biochemical properties. Here, we present a list of several software programs that use the information provided by water molecules to: i) analyse protein structures and provide the optimal positions of water molecules for protein hydration, ii) identify high-occupancy water sites in order to analyse ligand binding modes, and iii) detect and describe tunnels and cavities. The analysis of water molecules’ distribution and trajectories sheds a light on proteins’ interactions with small molecules, on the dynamics of tunnels and cavities, on protein composition and also on the functionality, transportation network and location of functionally relevant residues. Finally, the correct placement of water molecules in protein crystal structures can significantly improve the reliability of molecular dynamics simulations.

Structure–bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell.

Papaj, K.; Kasprzycka, A.; Góra, A.; Grajoszek, A.; Rzepecka, G.; Stojko, J.; Barski, J.-J.; Szeja, W.; Rusin, A. J Pharm Biomed Anal 2020, 185, 113216;https:// doi:10.1016/J.JPBA.2020.113216 ARTICLEOpen Access


The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram′-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative’s biological properties.



AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective.

Magdziarz T., Mitusińska K., Bzówka M., Raczyńska A., Stańczak A., Banas M., Bagrowska W., Góra A. ,Bioinformatics 2019, 1–3. ARTICLEOpen Access


Tunnels, pores, channels, pockets and cavities contribute to proteins architecture and performance. However, analysis and characteristics of transportation pathways and internal binding cavities are performed separately. We aimed to provide universal tool for analysis of proteins integral interior with access to detailed information on the ligands transportation phenomena and binding preferences.



Can a Mononuclear Iron(III)-Superoxo Active Site Catalyze the Decarboxylation of Dodecanoic Acid in UndA to Produce Biofuels?

Lin YT, Stańczak A, Manchev Y, Straganz GD, de Visser SP. Chemistry. 2019 Oct 4. ARTICLEOpen Access


Decarboxylation of fatty acids is an important reaction in cell metabolism, but also has potential in biotechnology for the biosynthesis of hydrocarbons as biofuels. The recently discovered nonheme iron decarboxylase UndA is involved in the biosynthesis of 1-undecene from dodecanoic acid and using X-ray crystallography was assigned to be a mononuclear iron species. However, the work was contradicted by spectroscopic studies that suggested UndA to be more likely a dinuclear iron system. To resolve this controversy we decided to pursue a computational study on the reaction mechanism of fatty acid decarboxylation by UndA using iron(III)-superoxo and diiron(IV)-dioxo models. We tested several models with different protonation states of active site residues. Overall, however, the calculations imply that mononuclear iron(III)-superoxo is a sluggish oxidant of hydrogen atom abstraction reactions in UndA and will not be able to activate fatty acid residues by decarboxylation at room temperature. By contrast, a diiron-dioxo complex reacts with much lower hydrogen atom abstraction barriers and hence is a more likely oxidant in UndA.



Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase.

Subramanian, K.; Mitusińska, K.; Raedts, J.; Almourfi, F.; Joosten, H.-J.; Hendriks, S.; Sedelnikova, S.E.; Kengen, S.W.M.; Hagen, W.R.; Góra, A.; Martins dos Santos, V.A.P.; Baker, P.J.; van der Oost, J.; Schaap, P.J., Biomolecules 2019, 9, 212. ARTICLEOpen Access


The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.



Reaction mechanism between Cu(II)-enolate complex and O2 as a test case for methodology used in DFT computational studies.

A. Stańczak, A.Miłaczewska, T. Borowski, T. J Mol Model (2019) 25: 122. ARTICLEOpen Access


The reaction mechanism of an intricate oxidation reaction of chlorodiketonate ligand of mononuclear Cu(II) complex was studied computationally employing five different models that differ in: a) basis set, b) the way that solvent corrections are included, and c) DFT functional. Qualitative and quantitative comparison of structures and enthalpy reaction profiles enabled us to assess how sensitive they are to the changes in computational methodology.




Exploring Solanum Tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking.

K. Mitusińska, T. Magdziarz, M. Bzówka, A. Stańczak, A. Góra, Biomolecules 2018, 8(4): 143. DOI: ARTICLEOpen Access


Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.



The modelling and enhancement of water hydrodynamics: general discussion.

Baaden, M.; Borthakur, M. P.; Casanova, S.; Coalson, R.; Freger, V.; Gonzalez, M.; Góra, A.; Hinds, B.; Hirunpinyopas, W.; Hummer, G.; Kumar, M.; Lynch, C.; Murail, S.; Noy, A.; Sansom, M.; Song, Q.; Vashisth, H.; Vögele, M., Faraday Discuss. 2018, 354–360, doi:10.1039/C8FD90021C. ARTICLE


Structure and function of natural proteins for water transport: General discussion.

Baaden, M.; Barboiu, M.; Bill, R. M.; Casanova, S.; Chen, C. L.; Conner, M.; Freger, V.; Gong, B.; Góra, A.; Hinds, B.; Horner, A.; Hummer, G.; Kumar, M.; Lokesh, M.; Mitra, S.; Noy, A.; Pohl, P.; Sadet, A.; Sansom, M.; Törnroth-Horsefield, S.; Vashisth, H., Faraday Discuss. 2018, 209, 83–95, doi:10.1039/C8FD90019A. ARTICLE



BALCONY: an R package for MSA and functional compartments of protein variability analysis.

A. Płuciennik, M. Stolarczyk, M. Bzówka, A. Raczyńska, T. Magdziarz, A. Góra, BMC Bioinformatics 2018, 19: 300. DOI:10.1186/s12859-018-2294-z. ARTICLEOpen Access


 Background: Here, we present an R package for entropy/variability analysis that facilitates prompt and convenient data extraction, manipulation and visualization of protein features from multiple sequence alignments. BALCONY can work with residues dispersed across a protein sequence and map them on the corresponding alignment of homologous protein sequences. Additionally, it provides several entropy and variability scores that indicate the conservation of each residue.

Results: Our package allows the user to visualize evolutionary variability by locating the positions most likely to vary and to assess mutation candidates in protein engineering.

Conclusion: In comparison to other R packages BALCONY allows conservation/variability analysis in context of protein structure with linkage of the appropriate metrics with physicochemical features of user choice.

Availability: CRAN project page: and our website: for major platforms: Linux/Unix, Windows and Mac OS X.



Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

K. Subramanian, A. Góra, R. Spruijt, K. Mitusińska, M. Suarez-Diez, V. M. dos Santos, P. J. Schaap,  PLoS ONE 13(6): e0198990. DOI: 10.1371/journal.pone.0198990 ARTICLEOpen Access



D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.





AQUA-DUCT a ligands tracking tool.

T. Magdziarz, K. Mitusińska, S. Gołdowska, A. Płuciennik, M. Stolarczyk, M .Ługowska and A. Góra; Bioinformatics (2017) 33 (13): 2045-2046. DOI: 10.1093/bioinformatics/btx125 ARTICLE or PROOF VERSIONOpen Access


Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.

Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.

Platform and Availability: Linux, Windows, macOS, OpenBSD,


The available copy of paper is a pre-copyedited, author-produced version of an article accepted for publication in Bioinformatics following peer review. The version of record Bioinformatics (2017) 33 (13): 2045-2046. is available online at: DOI: 10.1093/bioinformatics/btx125.



Engineering a de Novo Transport Tunnel ACS

J. Brezovsky, P. Babkova, O. Degtjarik, A. Fortova, A. Gora, I. Iermak, P. Rezacova, P. Dvorak, I. Smatanova, Z. Prokop, R. Chaloupkova, and J. Damborsky, Catalysis 2016, 6, 7597-7610 DOI: 10.1021/acscatal.6b02081 ARTICLE


Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members, up to 32-times increased substrate inhibition and 17-times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to the native enzyme, and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s-1 with 1,2-dibromoethane at 37oC and pH=8.6). Crystallographic analysis and molecular dynamics simulations confirmed successful introduction of structurally defined and functional transport tunnel. Our study demonstrates that whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels and we anticipate that this engineering strategy will facilitate creation of a wide range of useful biocatalysts.acs_catalysis_de_novo_tunnel


Molecular descriptor data explain market prices of a large commercial chemical compound library

J. Polanski, U. Kucia, R. Duszkiewicz, A. Kurczyk, T. Magdziarz and J. Gasteiger, Scientific Reports 6, Article number: 28521 (2016). doi:10.1038/srep28521 ARTICLEOpen Access


The relationship between the structure and a property of a chemical compound is an essential concept in chemistry srep28521-f1guiding, for example, drug design. Actually, however, we need economic considerations to fully understand the fate of drugs on the market. We are performing here for the first time the exploration of quantitative structure-economy relationships (QSER) for a large dataset of a commercial building block library of over 2.2 million chemicals. This investigation provided molecular statistics that shows that on average what we are paying for is the quantity of matter. On the other side, the influence of synthetic availability scores is also revealed. Finally, we are buying substances by looking at the molecular graphs or molecular formulas. Thus, those molecules that have a higher number of atoms look more attractive and are, on average, also more expensive. Our study shows how data binning could be used as an informative method when analyzing big data in chemistry.



New Publicly Available Chemical Query Language, CSRML, To Support Chemotype Representations for Application to Data Mining and Modeling

C. Yang, A. Tarkhov, J. Marusczyk, B. Bienfait, J. Gasteiger, T. Kleinoeder, T. Magdziarz, O. Sacher, C. H. Schwab, J. Schwoebel, L. Terfloth, K. Arvidson, A. Richard, A. Worth, and J. Rathman, J. Chem. Inf. Model., vol. 55, no. 3, pp. 510–528, Mar. 2015.


Chemotypes are a new approach for representing molecules, chemical substructures and patterns, reaction rules, and reactions. Chemotypes are capable of integrating types of information beyond what is possible usinYang_2015g current representation methods (e.g., SMARTS patterns) or reaction transformations (e.g., SMIRKS, reaction SMILES). Chemotypes are expressed in the XML-based Chemical Subgraphs and Reactions Markup Language (CSRML), and can be encoded not only with connectivity and topology but also with properties of atoms, bonds, electronic systems, or molecules. CSRML has been developed in parallel with a public set of chemotypes, i.e., the ToxPrint chemotypes, which are designed to provide excellent coverage of environmental, regulatory, and commercial-use chemical space, as well as t represent chemical patterns and properties especially relevant to various toxicity concerns. A software application, ChemoTyper has also been developed and made publicly available in order to enable chemotype searching and fingerprinting against a target structure set. The public ChemoTyper houses the ToxPrint chemotype CSRML dictionary, as well as reference implementation so that the query specifications may be adopted by other chemical structure knowledge systems. The full specifications of the XML-based CSRML standard used to express chemotypes are publicly available to facilitate and encourage the exchange of structural knowledge.


Integrative Modeling Strategies for Predicting Drug Toxicities at the eTOX Project

F. Sanz, P. Carrió, O. López, L. Capoferri, D. P. Kooi, N. P. E. Vermeulen, D. P. Geerke, F. Montanari, G. F. Ecker, C. H. Schwab, T. Kleinöder, T. Magdziarz, and M. Pastor, Mol. Inform., p. n/a–n/a, Jun. 2015.


Early prediction of safety issues in drug development is at the same time highly desirable and highly challenging. ReSanz_2015cent advances emphasize the importance of understanding the whole chain of causal events leading to observable toxic outcomes. Here we describe an integrative modeling strategy based on these ideas that guided the design of eTOXsys, the prediction system used by the eTOX project. Essentially, eTOXsys consists of a central server that marshals requests to a collection of independent prediction models and offers a single user interface to the whole system. Every of such model lives in a self-contained virtual machine easy to maintain and install. All models produce toxicity-relevant predictions on their own but the results of some can be further integrated and upgrade its scale, yielding in vivo toxicity predictions. Technical aspects related with model implementation, maintenance and documentation are also discussed here. Finally, the kind of models currently implemented in eTOXsys is illustrated presenting three example models making use of diverse methodology (3D-QSAR and decision trees, Molecular Dynamics simulations and Linear Interaction Energy theory, and fingerprint-based QSAR).



CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures

B. Kozlikova, E. Sebestova, V. Sustr, J. Brezovsky, O. Strnad, L. Daniel, D. Bednar, A. Pavelka, M. Manak, M. Bezdeka, P. Benes, M. Kotry, A. Gora, J. Damborsky, and J. Sochor, Bioinforma. Oxf. Engl., vol. 30, no. 18, pp. 2684–2685, Sep. 2014 ARTICLE.


 The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graphic user interface for setting up the calculation and interactive exploration of identified tunnels/channels and their characteristics.


Synthesis, antifeedant activity against Coleoptera and 3D QSAR study of alpha-asarone derivatives

B. Łozowicka, P. Kaczyński, T. Magdziarz, and A. T. Dubis, SAR QSAR Environ. Res., vol. 25, no. 3, pp. 173–188, Mar. 2014.


For the first time, a set of 56 compounds representing structural derivatives of naturally occurring alpha-asarone as an antifeedants against stored product pests Sitophilus granarius L., Trogoderma granarium Ev., and Tribolium confusum Duv., were subjected to the 3D QSAR studies. Three-dimensional quantitative structure–activity relationships (3D-QSAR) for 56 compounds, including 15 newly synthesized, were performed using comparative molecular field analysis s-CoMFA and SOM-CoMSA techniques. QSAR was conducted based on a combination of biological activity (against Coleoptera larvae and beetles) and various geometrical, topological, quantum-mechanical, electronic, and chromatographic descriptors. The CoMSA formalism coupled with IVE (CoMSA–IVE) allowed us to obtain highly predictive models for Trogoderma granarium Ev. larvae. We have found that this novel method indicates a clear molecular basis for activity and lipophilicity. This investigation will facilitate optimization of the design of new potential antifeedants.


Structure-Based Modeling of Dye-Fiber Affinity with SOM-4D-QSAR Paradigm: Application to Set of Anthraquinone Derivatives

A. Bak, M. Wyszomirski, T. Magdziarz, A. Smolinski, and J. Polanski, Comb. Chem. High Throughput Screen., vol. 17, no. 6, pp. 485–502, 2014.


A comparative structure-affinity study of anthraquinone dyes adsorption on cellulose fibre is presented in this paper. We used receptor-dependent 4D-QSAR methods based on grid and neural (SOM) methodology coupled with IVEPLS procedure. The applied RD 4D-QSAR approach focuses mainly on the ability of mapping dye properties to verify the concept of tinctophore in dye chemistry. Moreover, the stochastic SMV procedure to investigate the predictive ability of the method for a large population of 4D-QSAR models was employed. The obtained findings were compared with the previously published RI 3D/4D-QSAR models for the corresponding anthraquinone trainings sets. The neutral (protonated) and anionic (deprotonated) forms of anthraquinone scaffold were examined in order to deal with the uncertainty of the dye ionization state. The results are comparable to both the neutral and anionic dye sets regardless of the occupancy and charge descriptors applied, respectively. It is worth noting that the SOM-4D-QSAR behaves comparably to the cubic counterpart which is observed in each training/test subset specification (4D-QSAR-Jo vs SOM- 4D-QSARo and 4D-QSAR-Jq vs SOM-4D-QSARq). Additionally, an attempt was made to specify a common set of variables contributing significantly to dye-fiber binding affinity; it was simultaneously performed for some arbitrary chosen SMV models. The presented RD 4D-QSAR methodology together with IVE-PLS procedure provides a robust and predictive modeling technique, which facilitates detailed specification of the molecular motifs significantly contributing to the fiber-dye affinity.



Probing a Chemical Space for Fragmental Topology-Activity Landscapes (FRAGTAL): Application for Diketo Acid and Catechol HIV Integrase Inhibitor Offspring Fragments

A. Bak, T. Magdziarz, A. Kurczyk, K. Serafin, and J. Polanski, Comb. Chem. High Throughput Screen., vol. 16, no. 4, pp. 274–287, 2013.


Fragmental topology-activity landscapes (FRAGTAL), a new concept for encoding molecular descriptors for fragonomics into the framework of the molecular database records is presented in this paper. Thus, a structural repository containing biological activity data was searched in a substructure mode by a series of molecular fragments constructed in an incremental or decremental manner. The resulted series of database hits annotated with their activities construct FRAGTAL descriptors encoding a frequency of the certain fragments among active compounds and/or their activities. Actually, this method might be interpreted as a simplified adaptation of the frequent subgraph mining (FSM) method. The FRAGTAL method reconstructs the way in which medicinal chemists are used to designing a prospective drug structure intuitively. A representative example of the practical application of FRAGTAL within the ChemDB Anti-HIV/OI/TB database for disclosing new fragments for HIV-1 integrase inhibition is discussed. In particular, FRAGTAL method identifies ethyl malonate amide (EMA) as the diketo acid (DKA) related arrangement. Since new molecular constructs based on the EMA fragment are still a matter of future investigations we referred to this as anthe DKA offspring.


Gates of Enzymes

A. Gora, J. Brezovsky, and J. Damborsky, Chem. Rev., vol. 113, no. 8, pp. 5871–5923, 2013. ARTICLEOpen Access

Table of ContentsGATES_OF_ENZYMES

  1. Introduction
  2. Molecular Function of Gates
  3. Structural Basis of Gates
  4. Locations of Gates
  5. Engineering of Gates
  6. Conclusions


The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

K. Hasan, A. Gora, J. Brezovsky, R. Chaloupkova, H. Moskalikova, A. Fortova, Y. Nagata, J. Damborsky, and Z. Prokop, FEBS J., vol. 280, no. 13, pp. 3149–3159, July 2013.


FEBS2013 Haloalkane dehalogenases catalyse the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilising residues that are important in substrate binding and stabilisation of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenase, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 possesses a unique halide-stabilising tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the enzyme’s structure and catalytic properties were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen bonding patterns within the active sites of the wild-type and the mutant, and of the stabilisation of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding 3.7-fold, presumably due to the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the enzyme’s substrate specificity and reduced its K0.5 for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilises its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.


Software tools for identification, visualization and analysis of protein tunnels and channels

J. Brezovsky, E. Chovancova, A. Gora, A. Pavelka, L. Biedermannova, and J. Damborsky, Biotechnol. Adv., vol. 31, no. 1, pp. 38–49, Jan. 2013. ARTICLE


Protein structures contain highly complex systems of voids, making up specific features such as surface clefts or grooves, pockets, protrusions, cavities, pores or channels, and tunnels. Many of them are essential for the migration of solvents, ions and small molecules through proteins, and their binding to the functional sites. Analysis of these structural features is very important for understanding of structure-function relationships, for the design of potential inhibitors or proteins with improved functional properties. Here we critically review existing software tools specialized in rapid identification, visualization, analysis and design of protein tunnels and channels. The strengths and weaknesses of individual tools are reported together with examples of their applications for the analysis and engineering of various biological systems. This review can assist users with selecting a proper software tool for study of their biological problem as well as highlighting possible avenues for further development of existing tools. Development of novel descriptors representing not only geometry, but also electrostatics, hydrophobicity or dynamics, is needed for reliable identification of biologically relevant tunnels and channels.



Pharmacophore-based database mining for probing fragmental drug-likeness of diketo acid analogues

A. Bak, T. Magdziarz, and J. Polanski, SAR QSAR Environ. Res., vol. 23, no. 1–2, pp. 185–204, 2012.


A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB

L. Biedermannová, Z. Prokop, A. Gora, E. Chovancová, M. Kovács, J. Damborsky, and R. C. Wade, J. Biol. Chem., vol. 287, no. 34, pp. 29062–29074, Aug. 2012.ARTICLE



Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly JBC2012slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.


CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

E. Chovancova, A. Pavelka, P. Benes, O. Strnad, J. Brezovsky, B. Kozlikova, A. Gora, V. Sustr, M. Klvana, P. Medek, L. Biedermannova, J. Sochor, and J. Damborsky, PLoS Comput Biol, vol. 8, no. 10, p. e1002708, Oct. 2012.ARTICLEOpen Access


 Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures.

caver_mHerein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at


Quinoline scaffold as a privileged substructure in antimicrobial drugs

R. Musiol, T. Magdziarz, and A. Kurczyk, Sci. Microb. Pathog. Commun. Curr. Res. Technol. Adv. Badajoz Spain Formatex, pp. 72–83, 2011.


A. Bak, T. Magdziarz, A. Kurczyk, and J. Polanski, “Mapping fragmental drug-likeness in the MoStBioDat environment: intramolecular hydrogen bonding motifs in β-ketoenols,” Comb. Chem. High Throughput Screen., vol. 14, no. 7, pp. 560–569, 2011.


Mapping drug architecture by MoStBioDat: rapid screening of intramolecular hydrogen bonded motifs in catechols

A. Bak, T. Magdziarz, A. Kurczyk, and J. Polanski, Drug Dev. Res., vol. 72, no. 2, pp. 209–218, 2011.



Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

P. Mazur, T. Magdziarz, A. Bak, Z. Chilmonczyk, T. Kasprzycka-Guttman, I. Misiewicz-Krzemińska, K. Skupińska, and J. Polanski, J. Mol. Model., vol. 16, no. 7, pp. 1205–1212, 2010.



Receptor independent and receptor dependent CoMSA modeling with IVE-PLS: application to CBG benchmark steroids and reductase activators

T. Magdziarz, P. Mazur, and J. Polanski, J. Mol. Model., vol. 15, no. 1, pp. 41–51, 2009.


Mechanism of hydrogen abstraction by O- species in oxidative dehydrogenation of early alkanes: Propane, ethane and methane. Model theoretical DFT study

A. Gora and E. Broclawik, Pol. J. Chem., vol. 82, pp. 1779–1791, 2008.



Modeling robust QSAR.

J. Polanski, A. Bak, R. Gieleciak, and T. Magdziarz, J. Chem. Inf. Model., vol. 46, no. 6, pp. 2310–8, 2006.


T. Magdziarz, B. \Lozowicka, R. Gieleciak, A. Bąk, J. Polański, and Z. Chilmonczyk, “3D QSAR study of hypolipidemic asarones by comparative molecular surface analysis,” Bioorg. Med. Chem., vol. 14, no. 5, pp. 1630–1643, 2006.


Lower temperature dehydrogenation of methylcyclohexane by membrane-assisted equilibrium shift.

A. Gora, D. A. P. Tanaka, F. Mizukami, and T. M. Suzuki, Chem. Lett., vol. 35, pp. 1372–1373, 2006.ARTICLE


 Selective removal of hydrogen by the palladium membrane of novel configuration shifts the equilibrium in the dehydrogenation of methylcyclohexane allowing a continuous operation at below the critical temperature of palladium α–β phase transition.


Quantum chemical modeling of electrochromism of tungsten oxide films.

E. Broclawik, A. Gora, P. Liguzinski, P. Petelenz, and H. A. Witek, J. Chem. Phys., vol. 124, 2006.



Modeling Robust QSAR. 1. Coding Molecules in 3D-QSAR from a Point to Surface Sectors and Molecular Volumes.

R. Gieleciak, T. Magdziarz, A. Bak, and J. Polanski,J. Chem. Inf. Model., vol. 45, no. 5, pp. 1447–1455, Sep. 2005.


Quantum chemical modelling of the process of lithium insertion into WO(3) films

E. Broclawik, A. Gora, P. Liguzinski, P. Petelenz, and M. Slawik, Catal. Today, vol. 101, pp. 155–162, 2005. ARTICLE



GRID formalism for the comparative molecular surface analysis: application to the CoMFA benchmark steroids, azo dyes, and HEPT derivatives.

J. Polanski, R. Gieleciak, T. Magdziarz, and A. Bak, J. Chem. Inf. Comput. Sci., vol. 44, no. 4, pp. 1423–1435, 2004.


Self-organizing neural networks for Modeling robust 3D and 4D QSAR: Application to dihydrofolate reductase inhibitors.

J. Polanski, A. Bak, R. Gieleciak, and T. Magdziarz, Molecules, vol. 9, no. 12, pp. 1148–1159, 2004.


Analogues of the styrylquinoline and styrylquinazoline HIV-1 integrase inhibitors: design and synthetic problems.

J. Polański, H. Niedba\la, R. Musio\l, D. Tabak, B. Podeszwa, R. Gieleciak, A. Bak, A. Pa\lka, and T. Magdziarz, Acta Pol. Pharm., vol. 61, pp. 3–4, 2004.


A. Gora and E. Broclawik, “Theoretical estimation of acid-base properties of Lewis and Bronsted centres at the V-W-O catalyst surface: water molecule as the probe in DFT calculations,” J. Mol. Catal. -Chem., vol. 215, pp. 187–193, 2004.ARTICLE



Microscopic calculation of the static electric susceptibility of polyethylene.

A. Eilmes, R. W. Munn, V. G. Mavrantzas, D. N. Theodorou, and A. Gora, J. Chem. Phys., vol. 119, pp. 11458–11466, 2003.


Microscopic calculation of the energetics of ions in polyethylene.

A. Eilmes, R. W. Munn, and A. Gora, J. Chem. Phys., vol. 119, pp. 11467–11474, 2003.



The role of tungsten in formation of active sites for no SCR on the V-W-O catalyst surface – quantum chemical modeling (DFT).

E. Broclawik, A. Gora, and M. Najbar, J. Mol. Catal. -Chem., vol. 166, pp. 31–38, 2001.ARTICLE


Low-temperature reactivity of the surface species of vanadia-tungsta catalyst.

M. Najbar, A. Gora, A. Bialas, and A. Weselucha-Birczynska, Solid State Ion., vol. 141, pp. 499–506, 2001.ARTICLE



Quantum chemical modeling (DFT) of active species on the V-W-O catalyst surface in various redox conditions.

A. Gora, E. Broclawik, and M. Najbar, Comput. Chem., vol. 24, pp. 405–410, 2000.ARTICLE


Evolution of the surface species of the V2O5-WO3 catalysts.

M. Najbar, E. Broclawik, A. Gora, J. Camra, A. Bialas, and A. Weselucha-Birczynska, Chem. Phys. Lett., vol. 325, pp. 330–339, 2000.ARTICLE


Evolution of Ti-Sn-rutile-supported V2O5-WO3 catalyst during its use in nitric oxide reduction by ammonia.

M. Najbar, F. Mizukami, A. Bialas, J. Camra, A. Weselucha-Birczynska, H. Izutsu, and A. Gora, Top. Catal., vol. 11, pp. 131–138, 2000.ARTICLE


Gora, A., Brezovsky, J. & Damborsky, J., Computer-assisted enzyme engineering by modification of tunnels, channels and gates. Current Opinion in Biotechnology vol 22, supplement 1 September 2011.

Gora, A., Pacheco Tanaka, D. A., Mizukami, F. & Suzuki, T. M., Low temperature hydrogen recovering from organic storage compounds – application of pore fill type palladium membrane. Proceedings of International Conference and Exhibition on Green Chemistry, 18-21 IX 2006 Kuala Lumpur, Malaysia, 2006.

Gora, A. & Brocławik E. Dissociation of the Water Molecule on the V-W-O Catalyst Surface – Quantum Chemical Modeling. Polish Journal of Environmental Studies, 9(1), 31-34, 2000.

Najbar M., Białas A., Mizukami F., Wesełucha-Birczyńska A., Bielańska E. & Gora A. Vanadia-Tungsta DENOX Catalysts on High Surface Area Rutile. Polish Journal of Environmental Studies, 6, 83-8, 1997.


Prokop Z, Gora A, Brezovsky J, Chalupkova R, Stepankova V & Damborsky J Protein Engineering Handbook, Volume 3: Book chapter: Engineering of protein tunnels: Keyhole-lock-key model for catalysis by the enzymes with buried active sites – ISBN 978-3-527-33123-9 – Wiley-VCH, Weinheim.


US Patent No 13/604,094 “Method of thermostabilization of a protein and/or stabilization towards organic solvents” Jiri Damborsky, Zbynek Prokop, Tana Koudelakova, Veronika Stepankova, Radka Chaloupkova, Eva Chovancova, Artur Wiktor Gora, Jan Brezovsky.