Matched Peptides: Tuning Matched Molecular Pair Analysis for Biopharmaceutical Applications.

Biopharmaceuticals hold great promise for the future of drug discovery. Nevertheless, rational drug design strategies are mainly focused on the discovery of small synthetic molecules. Herein we present matched peptides, an innovative analysis technique for biological data related to peptide and protein sequences. It represents an extension of matched molecular pair analysis toward macromolecular sequence data and allows quantitative predictions of the effect of single amino acid substitutions on the basis of statistical data on known transformations. We demonstrate the application of matched peptides to a data set of major histocompatibility complex class II peptide ligands and discuss the trends captured with respect to classical quantitative structure-activity relationship approaches as well as structural aspects of the investigated protein-peptide interface. We expect our novel readily interpretable tool at the interface of cheminformatics and bioinformatics to support the rational design of biopharmaceuticals and give directions for further development of the presented methodology.

Identification of new potent GPR119 agonists by combining virtual screening and combinatorial chemistry.

Virtual screening in a huge collection of virtual combinatorial libraries has led to the identification of two new structural classes of GPR119 agonists with submicromolar in vitro potencies. Herein, we describe the virtual screening process involving feature trees fragment space searches followed by a 3D postprocessing step. The in silico findings were then filtered and prioritized, and finally, combinatorial libraries of target molecules were synthesized. Furthermore the so-called "activity-anchor principle" is introduced as an element to increase the chance to generate true hits. An activity anchor is a structural element expected to provide key contributions to a certain biological activity. Application of this technique has led to the discovery of two new GPR119-agonist hit series, one of which was further optimized to progress as a novel lead class.

Design of combinatorial libraries for the exploration of virtual hits from fragment space searches with LoFT.

A case study is presented illustrating the design of a focused CDK2 library. The scaffold of the library was detected by a feature trees search in a fragment space based on reactions from combinatorial chemistry. For the design the software LoFT (Library optimizer using Feature Trees) was used. The special feature called FTMatch was applied to restrict the parts of the queries where the reagents are permitted to match. This way a 3D scoring function could be simulated. Results were compared with alternative designs by GOLD docking and ROCS 3D alignments.

Minor groove binders and drugs targeting proteins cover complementary regions in chemical shape space.

DNA minor groove binders (MGBs) are known to influence gene expression and are therefore widely studied to explore their therapeutic potential. We identified shape-based virtual screening with ROCS as a highly effective computational approach to enrich known MGBs in top-ranked molecules. Discovery of ten previously unknown MGBs by shape-based screening further confirmed the relevance of ligand shape for minor groove affinity. Based on experimental testing we propose three simple rules (at least two positive charges, four nitrogen atoms, and one aromatic ring) as filters to reach even better enrichment of true positives in ROCS hit lists. Interestingly, shape-based ranking of MGBs versus FDA-approved drugs again leads to high enrichment rates, indicating complementary coverage of chemical shape space and indicating minor groove affinity to be unfavorable for approval of drugs targeting proteins.

SAR studies of non-zinc-chelating MMP-13 inhibitors: improving selectivity and metabolic stability.

SAR studies to improve the selectivity and metabolic stability of a class of recently discovered MMP-13 inhibitors are reported. Improved selectivity was achieved by modifying interactions with the S1' pocket. Metabolic stability was improved through reduction of inhibitor lipophilicity. This translated into lower in vivo clearance for the preferred compound.

Searching Fragment Spaces with feature trees.

Virtual combinatorial chemistry easily produces billions of compounds, for which conventional virtual screening cannot be performed even with the fastest methods available. An efficient solution for such a scenario is the generation of Fragment Spaces, which encode huge numbers of virtual compounds by their fragments/reagents and rules of how to combine them. Similarity-based searches can be performed in such spaces without ever fully enumerating all virtual products. Here we describe the generation of a huge Fragment Space encoding about 5 * 10(11) compounds based on established in-house synthesis protocols for combinatorial libraries, i.e., we encode practically evaluated combinatorial chemistry protocols in a machine readable form, rendering them accessible to in silico search methods. We show how such searches in this Fragment Space can be integrated as a first step in an overall workflow. It reduces the extremely huge number of virtual products by several orders of magnitude so that the resulting list of molecules becomes more manageable for further more elaborated and time-consuming analysis steps. Results of a case study are presented and discussed, which lead to some general conclusions for an efficient expansion of the chemical space to be screened in pharmaceutical companies.

Hydrogen-bonding patterns of minor groove-binder-DNA complexes reveal criteria for discovery of new scaffolds.

Minor groove-binding ligands are able to control gene expression and are of great interest for therapeutic applications. We extracted hydrogen-bonding geometries from all available structures of minor groove-binder-DNA complexes of two noncovalent binding modes, namely 1:1 (including hairpin and cyclic ligands) and 2:1 ligand/DNA binding. Positions of the ligand atoms involved in hydrogen bonding deviate from idealized hydrogen bond geometries and do not exploit the possibilities indicated by water molecules. Therefore, we suggest the inclusion of shape-based descriptors rather than hydrogen-bond patterns in virtual screening protocols for the identification of innovative minor groove-binding scaffolds.

Sequence-specific positions of water molecules at the interface between DNA and minor groove binders.

Ligands able to specifically recognize DNA sequences are of fundamental interest as transcription-controlling drugs. Herein, we analyze the positions of water molecules relative to B-DNA base pairs in the minor groove of X-ray and NMR protein data bank (PDB) structures. The patterns observed for water molecules at the interface between DNA and a ligand are compared with those obtained for structures without a ligand. Although the ligand end groups are often charged, and therefore highly hydrated, they do not alter the water patterns, which show considerable differences for the AT and CG base pairs. For AT they are much more precise than for CG in both ligand-containing and ligand-free structures. This behavior strongly indicates that the release of water molecules upon ligand binding leads to a gain of entropy and explains why this effect is especially pronounced for A-tract B-DNA sequences.

DNA minor groove pharmacophores describing sequence specific properties.

The more that is known about human and other genome sequences and the correlation between gene expression and the course of a disease, the more evident it seems to be that DNA is chosen as a drug target instead of proteins which are built with the information encoded by DNA. According to this approach, small minor groove binding molecules have been designed to bind the DNA sequence specifically and thereby downregulate genes. Because of their lack of druglikeness, we plan to use them as templates for forthcoming virtual screening experiments to discover molecules with the same bioactivity and a different scaffold. In this proof of principle study, carried out with the software tool Catalyst, we present a model work for description of a ligand-DNA complex with the aid of pharmacophore modeling methods. The successful reproduction of sequence specificity of a polyamidic minor groove binding ligand is the precondition for later model application to virtual screening.

Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment.

Autoimmune reactions to HSP60 are believed to play a key role during development of early atherosclerosis. Due to the high degree of phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for inducing cross-reactivity to self HSP60, which is expressed on the surface of arterial endothelial cells stressed by classical atherosclerosis risk factors. Conformational epitopes recognized by polyclonal anti-mycobacterial HSP60 antibodies from subjects with atherosclerosis were identified using a phage displayed random library of cyclic constrained 7mer peptides. After five rounds of selection, DNA sequencing of strongly binding clones revealed that one peptide motif (CIGSPSTNC) was present in 64% of all clones, and a second motif (CSFHYQNRC) in 14%. Using a newly developed method for structural alignment of small constrained peptides onto a protein surface, we located the motif present in 14% of all clones on the surface of mycobacterial HSP60. The motif present in 64% of all clones was found on the surface of mycobacterial HSP60 as well as in the homologous region of human HSP60, which makes this epitope a promising candidate for further investigations on cross-reactive epitopes involved in early atherogenesis.

Towards an understanding of DNA recognition by the methyl-CpG binding domain 1.

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be -3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.

Z-DNA's conformer substates revealed by FT-IR difference spectroscopy of nonoriented left-handed double helical poly(dG-dC).

Nonoriented hydrated films of double helical poly(dG-dC) in the Z-form were studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated slow-cooled samples between 290 and 220 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200, 220, and 240 K. IR spectral changes on isothermal relaxation at 200 and 220 K toward equilibrium, caused by interconversion of two conformer substates (CS) called Z1 and Z2, are revealed by IR difference spectra. Pronounced spectral changes on Z1-to-Z2 interconversion occur between approximately 750-1250 cm(-1) and these are attributed to structural changes of the phosphodiester-sugar backbone caused by changes of torsion angles, and to decreasing hydrogen-bonding of the ionic phosphate group with water molecules. These spectral changes on Z1-to-Z2 transition can be related to structural differences between ZI and ZII CS observed in single crystals. ZI/ZII CS occurs only at (dGpdC) base steps, and similar behavior is assumed for Z1/Z2. The Z1/Z2 population ratio was determined via curve resolution of marker bands for Z1 and Z2 centered at 785 and 779 cm(-1). This ratio is 0.64 at 290 K, corresponding to 39% of the phosphates of the (dGpdC) base steps in Z1 and 61% in Z2, and it increases to 1.24 on cooling to 220 K. For the Z2<=>Z1 equilibrium, an enthalpy change of -4.9 +/- 0.2 kJ mol(dGpdC)(-1) is obtained from the temperature dependence of the equilibrium constant. Z1 interconverts into Z2 at isothermal relaxation at 200 and 220 K, whereas on slow cooling from ambient temperature, Z2 interconverts into Z1. This unexpected reversal of CS interconversion is attributed to slow restructuring of hydration shells of the CS on quenching, in the same manner reported by Pichler et al. for the BI and BII CS of B-DNA (J. Phys. Chem. B 106, 3263-3274 (2002)). IR difference curves demonstrate two time scales on isothermal relaxation of Z1-->Z2 interconversion, a fast one for structural relaxation of the sugar-phosphate backbone, and a slow one for relaxation of the hydration shells. This slowing down of restructuring of CS hydration shells at approximately 220-240 K could be the cause for the suppression of biological functions at low temperatures.

The N6-methyl group of adenine further increases the BI stability of DNA compared to C5-methyl groups.

Methylated DNA bases are natural modifications which play an important role in protein-DNA interactions. Recent experimental and theoretical results have shown an influence of the base modification on the conformational behavior of the DNA backbone. MD simulations of four different B-DNA dodecamers (d(GC)(6), d(AT)(6), d(G(5mCG)(5)C), and d(A(T6mA)(5)T)) have been performed with the aim to examine the influence of methyl groups on the B-DNA backbone behavior. An additional control simulation of d(AU)(6) has also been performed to examine the further influence of the C5-methyl group in thymine. Methyl groups in the major groove (as in C5-methylcytosine, thymine, or N6-methyladenine) decrease the BII substate population of RpY steps. Due to methylation a clearer distinction of the BI substate stability between YpR and RpY (CpG/GpC or TpA/ApT) steps arises. A positive correlation between the BII substate population and base stacking distances is seen only for poly(GC). A methyl group added into the major groove increases mean water residence times around the purine N7 atom, which may stabilize the BI substate by improving the hydration network between the DNA backbone and the major groove. The N6-methyl group also forms a water molecule bridge between the N6 and O4 atoms, and thus further stabilizes the BI substate.

Cooperative effects on the formation of intercalation sites.

Daunomycin is one of the most important agents used in anticancer chemotherapy. It interacts with DNA through intercalation of its planar chromophore between successive base pairs. The effect of intercalation on structure, dynamics and energetics is the topic of a wealth of scientific studies. In the present study, we report a computational examination of the energetics of the intercalation process. In detail, we concentrate on the energetic penalty that intercalation of daunomycin introduces into DNA by disturbing it from its unbound conformation. For these means, we are analyzing already published molecular dynamics simulations of daunomycin-DNA complexes and present novel simulations of a bisdaunomycin-DNA and a 9-dehydroxydaunomycin-DNA intercalated complex using the MM-GBSA module implemented in the AMBER suite of programs. Using this molecular dynamics based, continuum solvent method we were able to calculate the energy required to form an intercalation site. Consequently, we compare the free energy of the duplex d(CGCGCGATCGCGCG)(2) in the B-form conformation with the respective conformations when intercalated with daunomycin and a bisintercalating analog. Our results show that the introduction of one single intercalation site costs approximately 32 kcal/mol. For double intercalation, or intercalation of the bisintercalator, the respective value for one intercalation site decreases to 27 and 24 kcal/mol, respectively, at a theoretical salt concentration of 0.15 M. This proposes that at least in these cases, a synergistic effect takes place. Although it is well known that intercalation leads to substantial disturbance of the DNA conformation, already performed investigations suggest a lower energetic penalty. Nevertheless to the best of our knowledge the calculations presented here are the most complete ones and consider hydration effects for the first time. The interaction energy between the ligand and the DNA certainly over-compensates this penalty for introducing the intercalation site and thus favors complexation. Such analyses are helpful for the description of allosteric effects in protein ligand binding.

Water-mediated contacts in the trp-repressor operator complex recognition process.

Water-mediated contacts are known as an important recognition tool in trp-repressor operator systems. One of these contacts involves two conserved base pairs (G(6).C(-6) and A(5). T(-5)) and three amino acids (Lys 72, Ile 79, and Ala 80). To investigate the nature of these contacts, we analyzed the X-ray structure (PDB code: 1TRO) of the trp-repressor operator complex by means of molecular dynamics simulations. This X-ray structure contains two dimers that exhibit structural differences. From these two different starting structures, two 10 ns molecular dynamics simulations have been performed. Both of our simulations show an increase of water molecules in the major groove at one side of the dimer, while the other side remains unchanged compared to the X-ray structure. Though the maximum residence time of the concerned water molecules decreases with an increase of solvent at the interface, these water molecules continue to play an important role in mediating DNA-protein contacts. This is shown by new stable amino acids-DNA distances and a long water residence time compared to free DNA simulation. To maintain stability of the new contacts, the preferential water binding site on O6(G6) is extended. This extension agrees with mutation experiment data on A5 and G6, which shows different relative affinity due to mutation on these bases [A. Joachimiak, T. E. Haran, P. B. Sigler, EMBO Journal 1994, Vol. 13, No. (2) pp. 367-372]. Due to the rearrangements in the system, the phosphate of the base G6 is able to interconvert to the B(II) substate, which is not observed on the other half side of the complex. The decrease of the number of hydrogen bonds between protein and DNA backbone could be the initial step of the dissociation process of the complex, or in other words an intermediate complex conformation of the association process. Thus, we surmise that these features show the importance of water-mediated contacts in the trp-repressor operator recognition process.

Daunomycin intercalation stabilizes distinct backbone conformations of DNA.

Daunomycin is a widely used antibiotic of the anthracycline family. In the present study we reveal the structural properties and important intercalator-DNA interactions by means of molecular dynamics. As most of the X-ray structures of DNA-daunomycin intercalated complexes are short hexamers or octamers of DNA with two drug molecules per doublehelix we calculated a self complementary 14-mer oligodeoxyribonucleotide duplex d(CGCGCGATCGCGCG)2 in the B-form with two putative intercalation sites at the 5'-CGA-3' step on both strands. Consequently we are able to look at the structure of a 1:1 complex and exclude crystal packing effects normally encountered in most of the X-ray crystallographic studies conducted so far. We performed different 10 to 20 ns long molecular dynamics simulations of the uncomplexed DNA structure, the DNA-daunomycin complex and a 1:2 complex of DNA-daunomycin where the two intercalator molecules are stacked into the two opposing 5'-CGA-3' steps. Thereby--in contrast to X-ray structures--a comparison of a complex of only one with a complex of two intercalators per doublehelix is possible. The chromophore of daunomycin is intercalated between the 5'-CG-3' bases while the daunosamine sugar moiety is placed in the minor groove. We observe a flexibility of the dihedral angle at the glycosidic bond, leading to three different positions of the ammonium group responsible for important contacts in the minor groove. Furthermore a distinct pattern of BI and BII around the intercalation site is induced and stabilized. This indicates a transfer of changes in the DNA geometry caused by intercalation to the DNA backbone.

3D-QSAR studies on natural acetylcholinesterase inhibitors of Sarcococca saligna by comparative molecular field analysis (CoMFA).

We have derived a comprehensive structure-activity relationship (SAR) picture for a new series of natural acetylcholinesterase inhibitors isolated from Sarcococca saligna. A set of 32 previously isolated and tested pregnane-type steroidal alkaloids inhibitors were investigated with respect to their IC(50) values (pIC(50)) against the AChE enzyme in order to derive CoMFA models using atom-based alignment. A highly significant CoMFA model was obtained with r(2) value of 0.974. The q(2) (cross validation r(2)) value also confirms the statistical significance of our model.

C5-methylation of cytosine in B-DNA thermodynamically and kinetically stabilizes BI.

We have examined the backbone dynamics of two alternating purine-pyrimidine dodecamers. One sequence consists of "pure" GC bases; the other one contains 5-methylcytosines. The effect of the methyl groups on the backbone substates BI/BII was investigated by means of molecular dynamics. The methylation influences, on one hand, the transition barrier between BI and BII and, on the other hand, the state of equilibrium. The kinetic consequences are an increase of the DeltaG of Gp5mC steps by 1.5 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol (compared with the nonmethylated DNA). Thus, the additive group differentiates between the two occurring dinucleotide steps and renders the phosphate of the 5-methylcytosine more rigid, as proposed by experimental studies. The thermodynamic consequences are an increase of the DeltaG of Gp5mC steps by 1.1 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol. The reason for this shift in equilibrium is still not completely clear on a molecular basis. But we can conclude that the indirect readout of DNA is influenced by methylation.

Molecular docking studies of natural cholinesterase-inhibiting steroidal alkaloids from Sarcococca saligna.

Alkaloids isolated from Sarcococca saligna significantly inhibit acetyl- and butyrylcholinesterase enzyme, suggesting discovery of inhibitors for nervous-system disorders. Studying interactions with the active site of the AChE enzyme from Torpedo californica, we have identified hydrophobic interactions inside the aromatic gorge area as the major stabilizing factor in enzyme-inhibitor complexes of these alkaloids. Molecular Dynamics simulation of a predicted complex indicates that ligand binding does not extensively alter enzyme structure, but reduces flexibility at the gorge.

Stepwise induced fit in the pico- to nanosecond time scale governs the complexation of the even-skipped transcriptional repressor homeodomain to DNA.

Induced fit effects in the complex of a DNA decamer with two even-skipped transcriptional repressor homeodomain molecules were investigated by means of molecular dynamics simulations. Dynamics of these effects are found to be in the time scale from pico- to nanoseconds. First steps are made by the fast-moving DNA backbone phosphates, which upon binding change their B(I)/B(II) substate distribution. Further rearrangements in the DNA double helix induced upon complexation, like bending of the helix axis, changes of the minor groove width, and of different helical parameters, are slower and occur within a few nanoseconds. The flexibility of the DNA, especially of its backbone, seems thereby to play an important role for specific DNA ligand recognition.