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The determination of k on and k off values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data of various aptamers. Upon plotting their k on and k off values as a function of their K d values, a notable trend emerged. Across a range of K d values spanning from 28 nM to 864 μM, the k on value decreased from 2×10 5  M -1  s -1 to 96 M -1  s -1 , whereas the k off value increased from 1.03×10 -3  s -1 to 0.012 s -1 . Thus, both k on and k off contributed to the change of K d in the same direction, although the range of k on change was larger. Since experiments are often run at close to the K d value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and offered insights into aptamer-based biosensors., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Protein-protein interactions (PPIs) are of utmost importance for maintenance of cellular homeostasis. Herein, a central role can be found for 14-3-3 proteins. These hub-proteins are known to bind hundreds of interaction partners, thereby regulating their activity, localization, and/or stabilization. Due to their ability to bind a large variety of client proteins, studies of 14-3-3 protein complexes flourished over the last decades, aiming to gain greater molecular understanding of these complexes and their role in health and disease. Because of their crucial role within the cell, 14-3-3 protein complexes are recognized as highly interesting therapeutic targets, encouraging the discovery of small molecule modulators of these PPIs. We discuss various examples of 14-3-3-mediated regulation of its binding partners on a mechanistic level, highlighting the versatile and multi-functional role of 14-3-3 within the cell. Furthermore, an overview is given on the development of stabilizers of 14-3-3 protein complexes, from initially used natural products to fragment-based approaches. These studies show the potential of 14-3-3 PPI stabilizers as novel agents in drug discovery and as tool compounds to gain greater molecular understanding of the role of 14-3-3-based protein regulation., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells., (© 2021 Wiley-VCH GmbH.)

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a K d of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation., (© 2021 Wiley-VCH GmbH.)

Proteasome activity is crucial for cell survival and proliferation. In recent years, small molecules have been discovered that can affect the catalytic activity of the proteasome. Rather than targeting the active sites of the proteasome, it might be possible to affect ubiquitin-dependent degradation of proteins by limiting the association of the 19S regulatory particle (19S RP) with the 20S core particle (20S CP) of the proteasome. We recently described the discovery of TXS-8, a peptoid that binds to Rpn-6. Rpn-6 is a proteasome-associated protein that makes critical contacts with the 19S RP and the 20S CP. Herein, we present a general workflow to evaluate the impact of a small-molecule binder on proteasome activity by using TXS-8 as an example. This workflow contains three steps in which specific probes or overexpressed proteins in cells are used to determine whether the hydrolysis activity of the proteasome is affected. Although, in our case, TXS-8 did not affect proteasome activity, our workflow is highly amenable to studying a variety of small-molecule-proteasome subunit interactions., (© 2021 Wiley-VCH GmbH.)

The effects of ligand binding on biomolecular conformation are crucial in drug design, enzyme mechanisms, the regulation of gene expression, and other biological processes. Descriptive models such as "lock and key", "induced fit", and "conformation selection" are common ways to interpret such interactions. Another historical model, linked equilibria, proposes that the free-energy landscape (FEL) is perturbed by the addition of ligand binding energy for the bound population of biomolecules. This principle leads to a unified, quantitative theory of ligand-induced conformation change, building upon the FEL concept. We call the map of binding free energy over biomolecular conformational space the "binding affinity landscape" (BAL). The perturbed FEL predicts/explains ligand-induced conformational changes conforming to all common descriptive models. We review recent experimental and computational studies that exemplify the perturbed FEL, with emphasis on RNA. This way of understanding ligand-induced conformation dynamics motivates new experimental and theoretical approaches to ligand design, structural biology and systems biology., (© 2020 Wiley-VCH GmbH.)

Regulator of G protein signaling (RGS) proteins have attracted attention as a result of their primary role in directing the specificity as well as the temporal and spatial aspects of G protein-coupled receptor signaling. In addition, alterations in RGS protein expression have been observed in a number of disease states, including certain cancers. In this area, RGS17 is of particular interest. It has been demonstrated that, while RGS17 is expressed primarily in the central nervous system, it has been found to be inappropriately expressed in lung, prostate, breast, cervical, and hepatocellular carcinomas. Overexpression of RGS17 leads to dysfunction in inhibitory G protein signaling and an overproduction of the intracellular second messenger cAMP, which in turn alters the transcription patterns of proteins known to promote various cancer types. Suppressing RGS17 expression with RNA interference (RNAi) has been found to decrease tumorigenesis and sufficiently prevents cancer cell migration, leading to the hypothesis that pharmacological blocking of RGS17 function could be useful in anticancer therapies. We have identified small-molecule fragments capable of binding the RGS homology (RH) domain of RGS17 by using a nuclear magnetic resonance fragment-based screening approach. By chemical shift mapping of the two-dimensional 15 N, 1 H heteronuclear single quantum coherence (HSQC) spectra of the backbone-assigned 15 N-labeled RGS17-RH, we determined the fragment binding sites to be distant from the Gα interface. Thus, our study identifies a putative fragment binding site on RGS17 that was previously unknown., (© 2021 Wiley-VCH GmbH.)

Probing the composition of the microbiome and its association with health and disease states is more accessible than ever due to the rise of affordable sequencing technology. Despite advances in our ability to identify members of symbiont communities, untangling the chemical signaling that they use to communicate with host organisms remains challenging. In order to gain a greater mechanistic understanding of how the microbiome impacts health, and how chemical ecology can be leveraged to advance small-molecule drug discovery from microorganisms, the principals governing communication between host and symbiont must be elucidated. Herein, we review common modes of interkingdom small-molecule communication in terrestrial and marine environments, describe the differences between these environments, and detail the advantages and disadvantages for studies focused on the marine environment. Finally, we propose the use of plant-endophyte interactions as a stepping stone to a greater understanding of similar interactions in marine invertebrates, and ultimately in humans., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our laboratory has recently identified two nanobodies (small antibodies produced by camelids)-Nb1 and Nb6-that bind efficiently to epithelial growth factor (EGF) and inhibit its ability to activate its receptor (EGFR). Because of the relevance of the EGF/EGFR axis as a target in oncology, these new nanobodies have promising therapeutic potential. This article, however, is focused on another feature of these nanobodies: their distinct thermodynamic signatures. Nb1 binds to EGF through an entropy-driven mechanism whereas Nb6 binds to this factor under enthalpic control. We discuss the advantages and disadvantages of each mechanism in the contexts of traditional medical chemistry (small-molecule drugs) and also of biological drugs. In this latter case, the implications in terms of selectivity are far from being clearly established and further experimental data are required. Their monomeric natures, high stability, and ease of recombinant production make nanobodies ideally suited for thermodynamic studies. Moreover, nanobodies, thanks to their simpler structures in comparison with conventional antibodies, might provide better understanding of the structural basis of the thermodynamic parameters of antigen recognition., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biological systems have evolved to create a structural and dynamic continuum of bio-macromolecular assemblies for the purpose of optimizing the system's functions. The formation of these dynamic higher-order assemblies is precisely controlled by biological cues. However, controlling the self-assembly of synthetic molecules spatiotemporally in or on live cells is still a big challenge, especially for performing functions. This concept article introduces the use of in situ reactions as a spatiotemporal control to form assemblies of small molecules that induce cell morphogenesis or apoptosis. After briefly introducing a representative example of a natural dynamic continuum of the higher-order assemblies, we describe enzyme-instructed self-assembly (EISA) for constructing dynamic assemblies of small molecules, then discuss the use of EISA for controlling cell morphogenesis and apoptosis. Finally, we provide a brief outlook to discuss the future perspective of this exciting new research direction., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10 4 copies per library member are used as the input., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The GACKIX activator binding domain has been a compelling target for small-molecule probe discovery because of the central role of activator-GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context-dependent function of activator-coactivator complexes. However, the dynamic and transient protein-protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules. An additional complication is that activator-binding motifs, such as GACKIX, are found in multiple coactivators, making specificity difficult to attain. In this study, we demonstrate that the strategy of tethering can be used to rapidly discover highly specific covalent modulators of the dynamic PPIs between activators and coactivators. These serve as both ortho- and allosteric modulators, enabling the tunable assembly or disassembly of the activator-coactivator complexes formed between the KIX domain and its cognate activator binding partners MLL and CREB. The molecules maintain their function and selectivity, even in human cell lysates and in bacterial cells, and thus, will ultimately be highly useful probes for cellular studies., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ride the wave! Biocatalysis uses nature's catalysts, enzymes and whole cell systems, for synthetic purposes. In a biotransformation, the biocatalyst transforms a well-defined substrate to the desired product, in contrast to the fermentation process, which produces the desired product from a complex mixture of nutrients. Biocatalysis has reached an industrially established level through several waves of technological evolution; participants of the BioTrans 2017 conference in Budapest could witness the newest wave of this technology., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap 3 A) are important factors in cancer development and progression. Fhit has Ap 3 A hydrolase activity and cleaves Ap 3 A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit-mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit⋅Ap 3 A complex might exert its growth-suppressive function remain to be discovered. Small-molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor-suppressive functions. Here we describe the development of a high-throughput screen for the identification of such small-molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC 50 values in the nanomolar range., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phage-display libraries and DNA-encoded chemical libraries (DECLs) represent useful tools for the isolation of specific binding molecules from large combinatorial sets of compounds. With both methods, specific binders are recovered at the end of affinity capture procedures by using target proteins of interest immobilized on a solid support. However, although the efficiency of phage-display selections is routinely quantified by counting the phage titer before and after the affinity capture step, no similar quantification procedures have been reported for the characterization of DECL selections. In this article, we describe the potential and limitations of quantitative PCR (qPCR) methods for the evaluation of selection efficiency by using a combinatorial chemical library with more than 35 million compounds. In the experimental conditions chosen for the selections, a quantification of DNA input/recovery over five orders of magnitude could be performed, revealing a successful enrichment of abundant binders, which could be confirmed by DNA sequencing. qPCR provided rapid information about the performance of selections, thus facilitating the optimization of experimental conditions., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The binding of the scaffolding protein MO25 to SPAK and OSR1 protein kinases, which regulate ion homeostasis, causes increases of up to 100-fold in their catalytic activity. Various animal models have shown that the inhibition of SPAK and OSR1 lowers blood pressure, and so here we present a new indirect approach to inhibiting SPAK and OSR1 kinases by targeting their protein partner MO25. To explore this approach, we developed a fluorescent polarisation assay and used it in screening of a small in-house library of ≈4000 compounds. This led to the identification of one compound-HK01-as the first small-molecule inhibitor of the MO25-dependent activation of SPAK and OSR1 in vitro. Our data confirm the feasibility of targeting this protein-protein interaction by small-molecule compounds and highlights their potential to modulate ion co-transporters and thus cellular electrolyte balance., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The prolyl hydroxylase domain (PHD) protein:hypoxia inducible factor (HIF) pathway is the main pathway by which changes in oxygen concentration are transduced to changes in gene expression. In mammals, there are three PHD paralogues, and PHD2 has emerged as a particularly critical one for regulating HIF target genes such as erythropoietin (EPO), which controls red cell mass and hematocrit. PHD2 is distinctive among the three PHDs in that it contains an N-terminal MYND-type zinc finger. We have proposed that this zinc finger binds a Pro-Xaa-Leu-Glu (PXLE) motif found in proteins of the HSP90 pathway to facilitate HIF-α hydroxylation. Targeting this motif could provide a means of specifically inhibiting this PHD isoform. Here, we screened a library of chemical compounds for their capacity to inhibit the zinc finger of PHD2. We identified compounds that, in vitro, can inhibit PHD2 binding to a PXLE-containing peptide and induce activation of HIF. Injection of one of these compounds into mice induces an increase in hematocrit. This study offers proof of principle that inhibition of the zinc finger of PHD2 can provide a means of selectively targeting PHD2 to activate the HIF pathway., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pseudomonas aeruginosa uses N-acylated L-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins--LasR, RhlR, and QscR--for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression and, thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered to be at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding that of LasR. Herein, we report the characterization of a set of synthetic, small-molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrated that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Energy-coupling factor (ECF) transporters are membrane-protein complexes that mediate vitamin uptake in prokaryotes. They bind the substrate through the action of a specific integral membrane subunit (S-component) and power transport by hydrolysis of ATP in the three-subunit ECF module. Here, we have studied the binding of thiamine derivatives to ThiT, a thiamine-specific S-component. We designed and synthesized derivatives of thiamine that bind to ThiT with high affinity; this allowed us to evaluate the contribution of the functional groups to the binding affinity. We determined six crystal structures of ThiT in complex with our derivatives. The structure of the substrate-binding site in ThiT remains almost unchanged despite substantial differences in affinity. This work indicates that the structural organization of the binding site is robust and suggests that substrate release, which is required for transport, requires additional changes in conformation in ThiT that might be imposed by the ECF module., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cytochrome P450 monooxygenases (P450s) are versatile enzymes with high potential for biocatalysis. The number of newly annotated P450 genes has been increasing constantly, and these thus represent a rich resource for new biocatalysts. However, the substrate scopes of newly identified P450s are often not known, and thus their exploitation is difficult. Herein we describe an approach, named "cluster screening", and its application for the primary characterization of two P450s: CYP154E1 and CYP154A8. A library comprising 51 compounds was designed and organized into nine groups according to their chemical properties. The activities of both P450s in vitro were maintained with suitable nonphysiological redox partners, and the cluster library was screened with these enzymes for product formation. From this library, 30 compounds tested positive for CYP154E1 and 23 were positive for CYP154A8. Cluster screening distinguishes subtle differences in activity and selectivity of enzymes as closely related as those of the same P450 family. For example, the alkaloid pergolide mesylate was converted by CYP154E1 (4 %) but not by CYP154A8. A building block of vitamin D3 , Grundmann's ketone, was converted by both enzymes, although conversion was higher with CYP154E1 (100 vs 53%)., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PHOTO OPPORTUNITY: We have developed a dual photoaffinity labeling system in which an active and an inactive probe bearing orthogonal detection groups are co-reacted in a single photoreaction. The approach allowed selective fluorescent detection of a model binding protein in cell lysate by either 1D or 2D electrophoresis., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability to identify inhibitors of protein-protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein-protein interaction site., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. The use of TS inhibitors in cancer chemotherapy suffers from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein-binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS-binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme-targeting drugs to overcome resistance development during chemotherapy., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Not without a chaperone: Pharmacological chaperones are designed to bind and ideally stabilise their target protein. Here, we elucidate the molecular mechanism of a potential pharmacological chaperone to treat phenylketonuria. The crystal structure of human phenylalanine hydroxylase with compound IV may help in the rational design of more efficient compounds to treat this disease., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nature constructs intricate complexes containing numerous binding partners in order to direct a variety of cellular processes. Researchers have taken a cue from these events to develop synthetic molecules that can nucleate natural and unnatural interactions for a diverse set of applications. These molecules can be designed to drive protein dimerization or to modulate the interactions between proteins, lipids, DNA, or RNA and thereby alter cellular pathways. A variety of components within the cellular machinery can be recruited with or replaced by synthetic compounds. Directing the formation of multicomponent complexes with new synthetic molecules can allow unprecedented control over the cellular machinery.

RNA interference is triggered by small hairpin precursors that are processed by the endonuclease dicer to yield active species such as siRNAs and miRNAs. To regulate the RNAi-mediated suppression of gene expression, we imagined a strategy that relies on the sequence-specific inhibition of shRNA precursor processing by immediate RNA-small molecule interactions. Here, we present a first step in this direction by augmenting shRNAs with guanosine-rich sequences that are prone to fold into four-stranded structures. The addition of small molecules that selectively bind to such quadruplex sequences should allow for the specific inhibition of dicing of shRNAs that contain suitable G-rich elements. In an attempt to find compounds that protect against dicer processing, we have examined the effects of quadruplex-binding compounds on the dicer processing of shRNAs containing G-quadruplexes. Although a variety of small molecules that are known to bind to quadruplexes inhibited in vitro dicing of shRNAs, only two substance classes, namely certain porphyrazines and bisquinolinium compounds, showed selective inhibition of G-rich shRNAs compared to control sequences lacking guanine-rich elements. The G-rich shRNAs displayed a potent knockdown of gene expression in mammalian cell culture, but the effect was not influenced by addition of the respective quadruplex-binding compounds.