Defining the mobility range of a hinge-type connection using molecular dynamics and metadynamics

Plos One 2020 15(4):e0230962 Philip Horx, Armin Geyer


Supporting Information


Abstract A designed disulfide-rich β-hairpin peptide that dimerizes spontaneously served as a hinge-type connection between proteins. Here, we analyze the range of dynamics of this hinge dimer with the aim of proposing new applications for the DNA-encodable peptide and establishing guidelines for the computational analysis of other disulfide hinges. A recent structural analysis based on nuclear magnetic resonance spectroscopy and ion mobility spectrometry revealed an averaged conformation in the hinge region which motivated us to investigate the dynamic behavior using a combination of molecular dynamics simulation, metadynamics and free energy surface analysis to characterize the conformational space available to the hinge. Principal component analysis uncovered two slow modes of the peptide, namely, the opening and closing motion and twisting of the two β-hairpins assembling the hinge. Applying a collective variable (CV) that mimics the first dominating mode, led to a major expansion of the conformational space. The description of the dynamics could be achieved by analysis of the opening angle and the twisting of the β-hairpins and, thus, offers a methodology that can also be transferred to other derivatives. It has been demonstrated that the hinge peptide’s lowest energy conformation consists of a large opening angle and strong twist but is separated by small energy barriers and can, thus, adopt a closed and untwisted structure. With the aim of proposing further applications for the hinge peptide, we simulated its behavior in the sterically congested environment of a four-helix bundle. Preliminary investigations show that one helix is pushed out and a three-helix bundle forms. The insights gained into the dynamics of the tetra-disulfide peptide and analytical guidelines developed in this study may contribute to the understanding of the structure and function of more complex hinge-type proteins, such as the IgG antibody family.





Comparing the hinge-type mobility of natural and designed intermolecular bi-disulfide domains

Front. Chem. 2020, 8, 25 Philip Horx, Armin Geyer

DOI: 10.3389/fchem.2020.00025

Supporting Information


Abstract A pair of intermolecular disulfide bonds connecting two protein domains restricts their relative mobility in a systematic way. The bi-disulfide hinge cannot rotate like a single intermolecular disulfide bond yet is less restrained than three or more intermolecular disulfides which restrict the relative motion to a minimum. The intermediate mobility of bi-disulfide linked domains is characterized by their dominating opening and closing modes comparable to the mechanics of a door hinge on the macroscopic scale. Here we compare the central hinge region of Immunoglobulin G1 (IgG1) which is highly conserved among different species, with a recently designed hinge-type motif CHWECRGCRLVC from our lab, that was successfully used for the dimerization of the IgG1/κ-ab CL4 monocolonal antibody (mab). The minimal length of these synthetic hinges comprises only 12 amino acids, rendering them ideal models for computational studies. Well-tempered metadynamics was performed to adequately describe the available conformational space defined by the different hinges. In spite of the differences in amino acid composition and ring sizes, there are characteristic similarities of designed and natural hinges like the dependent mobility of the individual strands of each hinge domain.


Graphical abstract: The role of phosphopeptides in the mineralisation of silica


The role of phosphopeptides in the biomineralization of silica

Org. Biomol. Chem. 2020, 18, 700-706 Fabian Daus, Erik Pfeifer, Kevin Seipp, Norbert Hampp, Armin Geyer

DOI: 10.1039/c9ob02438g

Supporting Information


Abstract We investigated the silicification activity of hyperphosphorylated peptides in combination with long-chain polyamines (LCPA). The bioinspired in vitro silicification experiments with peptides containing different amounts of phosphorylated serines showed structure–activity dependence by altering the amount and morphology of the silica precipitate. Our study provides an explanation for the considerable metabolic role of diatoms in the synthesis of hyperphosphorylated poly-cationic peptides such as natSil-1A1. The efficient late-stage phosphorylation of peptides yielded a synthetic heptaphosphopeptide whose silicification properties resemble those of natSil-1A1. As opposed to this, unphosphorylated poly-cationic peptides or LCPA require concentrations above 1 mM for silicification. Hyperphosphorylated peptides showed a linear dependence between the amount of dissolved peptides and the amount of precipitated silica in the concentration range below 1 mM. Under mildly acidic conditions and short precipitation times, the concentration of the added LCPA determined the size of the silica spheres.





The synthesis, characterization, cytotoxic activity assessment and structure–activity relationship of 4-aryl-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitriles

Molecules 2019, 24, 4072-4083 Mahmoud Al-Refai, Mohammad M. Ibrahim, Mohamad Nurul Azmi, Hasnah Osman, Mohamad Hafizi Abu Bakar, Armin Geyer

DOI: 10.3390/molecules24224072

Supplementary Material


Abstract A series of 2-methoxypyridine-3-carbonitrile (5ai)-bearing aryl substituents were successfully synthesized in good yields by the condensation of chalcones (4ai) with malononitrile in basic medium. The condensation process, in most cases, offers a route to a variety of methoxypyridine derivatives (6ag) as side products in poor yields. All new compounds were fully characterized using different spectroscopic methods. Mass ESI-HMRS measurements were also performed. Furthermore, these compounds were screened for their in vitro cytotoxicity activities against three cancer cell lines; namely, those of the liver (line HepG2), prostate (line DU145) and breast (line MBA-MB-231). The cytotoxicity assessment revealed that compounds 5d, 5g, 5h and 5i exhibit promising antiproliferative effects (IC50 1–5 µM) against those three cancer cell lines.





Reversible covalent end-capping of collagen model peptides

Chem. Eur. J. 2019, 25, 14278-14283 Christoph Priem, Armin Geyer

DOI: org/10.1002/chem.201903460

Supporting Information


Abstract The combination of supramolecular aggregation of collagen model peptides with reversible covalent end‐capping of the formed triple helix in a single experimental set‐up yielded minicollagens, which were characterized by a single melting temperature. In spite of the numerous possible reaction intermediates, a specific synthetic collagen with a leading, middle and trailing strand is formed in a highly cooperative self‐assembly process.



Graphical abstract: Directed C(sp3)–H arylation of tryptophan: transformation of the directing group into an activated amide


Directed C(sp3)−H arylation of tryptophan: transformation of the directing group into an activated amide 

Chem. Sci. 2019, 10, 8634-8641. Lennart Nicke, Philip Horx, Klaus Harms, Armin Geyer

DOI: 10.1039/c9sc03440d

Supporting Information


Abstract The he 8-aminoquinoline (8AQ) directed C(sp3)–H functionalization was applied in the synthesis of β-arylated tryptophan derivatives. The laborious protecting group reorganization towards α-amino acids compatible for solid phase peptide synthesis (SPPS) was cut short by the transformation of the directing group into an activated amide, which was either used directly in peptide coupling or in the gram scale synthesis of storable Fmoc-protected amino acids for SPPS. In this work, directed C–H activation and nonplanar amide chemistry complement each other for the synthesis of hybrids between phenylalanine and tryptophan with restricted side chain mobility.




Side chain orientation of tryptophan analogs determines agonism and inverse agonism in short ghrelin peptides

ChemMedChem 2019, 14, 1849-1855. Lennart Nicke, Ronny Müller, Armin Geyer, Sylvia Els‐Heindl

DOI: 10.1002/cmdc.201900409

Supporting Information


Abstract We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe4 is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL‐NH2, the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid‐phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.



[Scheme 1]


Synthesis, characterization, crystal structure and supramolecularity of ethyl (E)-2-cyano-3-(3-methylthiophen-2-yl)acrylate and a new polymorph of ethyl (E)-2-cyano-3-(thiophen-2-yl)acrylate

Acta Cryst. 2019, E75, 1357-1361. Mahmoud Al-Refai, Basem F. Ali, Ala´a B. Said, Armin Geyer, Michael Marsch, Klaus Harms

DOI: 10.1107/S2056989019011435

Supporting Information


Abstract The synthesis, crystal structure and structural motif of two thio­phene-based cyano­acrylate derivatives, namely, ethyl (E)-2-cyano-3-(3-methyl­thio­phen-2-yl)acrylate (1), C11H11NO2S, and ethyl (E)-2-cyano-3-(thio­phen-2-yl)acrylate (2), C10H9NO2S, are reported. Derivative 1 crystallized with two independent molecules in the asymmetric unit, and derivative 2 represents a new monoclinic (C2/m) polymorph. The mol­ecular conformations of 1 and the two polymorphs of 2 are very similar, as all non-H atoms are planar except for the methyl of the ethyl groups. The inter­molecular inter­actions and crystal packing of 1 and 2 are described and compared with that of the reported monoclinic (C2/m) polymorph of derivative 2 [Castro Agudelo et al. (2017). Acta Cryst. E73, 1287–1289].




[Scheme 1]



IUCrData 2019, 4, x191046.  Mohammad M. Ibrahim, Mahmoud Al-Refai, Basem F. Ali, Armin Geyer, Klaus Harms, Michael Marsch

DOI: org/10.1107/S2414314619010460

Supporting Information


Abstract In the title compound, C16H16Cl2N2O2S, the pyrazole ring has an envelope conformation with the C atom bearing the phenyl ring being the flap. The dihedral angles between the central pyrazole ring (all atoms) and pendant thio­phene and phenyl rings are 2.00 (14) and 81.49 (12)°, respectively. In the crystal, weak C—H...O, Cl...π and π–π stacking inter­actions link the mol­ecules into a three-dimensional network.






Synthesis, characterization and cytotoxicity of new nicotinonitriles and their furo[2,3-b]pyridine derivatives.

J. Iran. Chem. Soc. 2019, 16, 715-722. Mohammad M. Ibrahim, Mahmoud Al-Refai, Mohamad Nurul Azmi, Hasnah Osman, Mohamad Hafizi Abu Bakar, Armin Geyer

DOI: org/10.1007/s13738-018-1549-y

Supporting Information


Abstract The present research work describes the synthesis of new series of nicotinonitrile (2) and furo[2,3-b]pyridine (3) heterocycles bearing thiophene substituent. The nicotinonitrile derivatives were prepared from the corresponding 3-cyano-(2H)-pyridones (1af) in excellent yields. The ring cyclization of the nicotinonitrile derivatives (2af) in the presence of sodium methoxide provided the furo[2,3-b]pyridines (3a–f) in moderate to good yields. All the newly synthesized compounds were characterized by extensive NMR analysis data, including 1D-NMR experiments (1H and 13C) and 2D-NMR experiments (COSY, HMBC, HSQC), as well as HRESIMS data. All the final products were subjected for cytotoxic activity against five different tumour cell lines including HeLa (cervical), DU145 (prostate), HepG2 (liver), MDA-MB-231 (breast-ER negative) and MCF7 (breast-ER positive), in addition to HSF1184 (normal human fibroblast) using the MTT assay. Compounds 2d and 3e were found to exhibit promising cytotoxicity with IC50 of < 20 µM against all different tested tumour cell lines. In addition, these compounds showed high selectivity (40–287 folds) for tumour cells over normal cells.




Diamondoid Amino Acid-based Peptide Kinase A Inhibitors

ChemMedChem 2019, 14, 663-672. Janis Müller, Romina A. Kirschner, Jan‐Philipp Berndt, Tobias Wulsdorf, Alexander Metz, Radim Hrdina, Peter R. Schreiner, Armin Geyer, Gerhard Klebe

DOI: 10.1002/cmdc.201800779

Supporting Information


Abstract The incorporation of diamondoid amino acids (DAAs) into peptide‐like drugs is a general strategy to improve lipophilicity, membrane permeability, and metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA‐containing kinase inhibitors of protein kinase A using a sophisticated molecular dynamics protocol and solid‐phase peptide synthesis. By means of a thermophoresis binding assay, NMR, and crystal structure analysis, we determined the influence of the DAAs on the secondary structure and binding affinity in comparison to the native protein kinase inhibitor, which is purely composed of proteinogenic amino acids. Affinity and binding pose are largely conserved. One variant showed 6.5‐fold potency improvement, most likely related to its increased side chain lipophilicity. A second variant exhibited slightly decreased affinity presumably due to loss of hydrogen‐bond contacts to surrounding water molecules of the first solvation shell.


Figure 1

Conceptional design of self-assembling bisubstrate-like inhibitors of protein kinase a resulting in a boronic acid glutamate linkage

ACS Omega 2019, 4, 775-784. Janis Müller, Romina A. Kirschner, Armin Geyer, Gerhard Klebe

DOI: 10.1021/acsomega.8b02364

Supporting Information


Abstract The spontaneous esterification of boronic acids with polyols provides a promising opportunity to generate self-assembled bisubstrate-like inhibitors within the binding pocket of cAMP-dependent protein kinase (PKA). As a first substrate component, we designed amino acids, which have either a boronic acid or ribopyranose side chain and introduced them to the substrate-like peptide protein kinase inhibitor (PKI). The second component was derived from the active-site inhibitor Fasudil, which was functionalized with phenylboronic acid. NMR spectroscopy in dimethylsulfoxide proved spontaneous reversible condensation of both components. Reinforced by the protein environment, both separately bound substrates were expected to react via boronic-ester formation bridging the two binding sites of PKA. Multiple crystal structures of PKA with bound PKIs, positionally modified with residues such as a ribopyranosylated serine and threonine or a phenylboronic acid attached to lysine via amide bonds, were determined with the phenylboronic acid-linked Fasudil. Although PKA accepts both inhibitors simultaneously, the expected covalent attachment between both components was not observed. Instead, spontaneous reaction of the terminal boronic acid group of the modified Fasudil with the carboxylate of Glu127 was detected once the latter residue is set free from a strong salt bridge formed with arginine by the original peptide inhibitor PKI. Thus, the desired self-assembly reaction occurs spontaneously in the protein environment by an unexpected carboxylic acid boronate complex. To succeed with our planned self-assembly reaction between both substrate components, we have to redesign the required reaction partners more carefully to finally yield the desired bisubstrate-like inhibitors in the protein environment.