Alexander Wolf

Identification and Structure of Small-Molecule Stabilizers of 14-3-3 Protein-Protein Interactions

Two structurally unrelated small molecules that stabilize the interaction of a 14–3–3 protein with the proton pump PMA2 have been identified. The compounds are selective among different 14–3–3 protein–protein interactions and are active in vivo. Crystal structures of ternary complexes revealed that the molecules bind to different sites in the interface of the 14–3–3 protein and PMA2 (see picture), thus explaining the different binding kinetics.

High-Throughput Screening To Identify Inhibitors Which Stabilize Inactive Kinase Conformations in p38α

Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.

Strategies to search and design stabilizers of protein–protein interactions: A feasibility study

Since protein–protein interactions play a pivotal role in the communication on the molecular level in virtually every biological system and process, the search and design for modulators of such interactions is of utmost importance. In recent years many inhibitors for specific protein–protein interactions have been developed, however, in only a few cases, small and druglike molecules are able to interfere in the complex formation of proteins. On the other hand, there are several small molecules known to modulate protein–protein interactions by means of stabilizing an already assembled complex. To achieve this goal, a ligand is binding to a pocket, which is located rim‐exposed at the interface of the interacting proteins, for example as the phytotoxin Fusicoccin, which stabilizes the interaction of plant H+‐ATPase and 14‐3‐3 protein by nearly a factor of 100. To suggest alternative leads, we performed a virtual screening campaign to discover new molecules putatively stabilizing this complex. Furthermore, we screen a dataset of 198 transient recognition protein–protein complexes for cavities, which are located rim‐exposed at their interfaces. We provide evidence for high similarity between such rim‐exposed cavities and usual ligands accommodating active sites of enzymes. This analysis suggests that rim‐exposed cavities at protein–protein interfaces are druggable binding sites. Therefore, the principle of stabilizing protein–protein interactions seems to be a promising alternative to the approach of the competitive inhibition of such interactions by small molecules. Proteins 2007.

Psoromic Acid is a Selective and Covalent Rab-Prenylation Inhibitor Targeting Autoinhibited RabGGTase

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 μM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.

A Novel CDK7 Inhibitor of the Pyrazolotriazine Class Exerts Broad-Spectrum Antiviral Activity at Nanomolar Concentrations

ABSTRACT Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.

Development of Selective, Potent RabGGTase Inhibitors

Members of the Ras superfamily of small GTPases are frequently mutated in cancer. Therefore, inhibitors have been developed to address the acitivity of these GTPases by inhibiting their prenylating enzymes FTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I, only a handful of RabGGTase inhibitors have been developed. The most active RabGGTase inhibitor known until recently was an FTase inhibitor which hit RabGGTase as an off-target. We recently reported our efforts to tune the selectivity of these inhibitors toward RabGGTase. Here we describe an extended set of selective inhibitors. The requirements for selective RabGGTase inhibitors are described in detail, guided by multiple crystal structures. In order to relate in vitro and cellular activity, a high-throughput assay system to detect the attachment of [(3)H]geranylgeranyl groups to Rab was used. Selective RabGGTase inhibition allows the establishment of novel drug discovery programs aimed at the development of anticancer therapeutics.

The Ras Pathway Modulator Melophlin A Targets Dynamins

The Ras/mitogen-activated protein (MAP) kinase signal transduction pathway regulates numerous biological programs including cell growth and differentiation, and harbors several important anticancer-drug targets. Recent research, in particular inspired by systems biology approaches, revealed the importance of dynamic spatiotemporal regulation of and interplay between the Ras network members and their interaction with other signaling modules for fully functional Ras signaling. Because of their rapid, conditional, and reversible mode of action, small-molecule modulators of protein function are particularly suitable tools for the conditional analysis of such dynamic biological processes, and hold great promise for the study of biological systems. Therefore, the identification of novel small-molecule modulators of signaling through the Ras network and the identification of their molecular targets are of major interest. 6] The naturally occurring tetramic acids melophlin A and B (1 and 2, Scheme 1A) reverse the morphology of HRas-transformed NIH3T3 fibroblasts at a concentration of 5 mgmL 1 (that is, IC50= 14 mm). [7] However, the biological targets of the melophlins and their link to the Ras network have not been identified. Herein, we report the synthesis of a melophlin-inspired compound collection and a subsequent chemical proteomics investigation, which revealed that melo-

Evaluation of Time Dependence and Interindividual Differences in Benzo[a]pyrene-Mediated CYP1A1 Induction and Genotoxicity in Porcine Urinary Bladder Cell Cultures∗

Exposure to tobacco smoke is an established cause of cancer in humans and cigarette smoking is a risk factor for urinary bladder cancer development. Aromatic amines are believed responsible for the bladder-specific carcinogenic effect, but polycyclic aromatic hydrocarbons (PAHs) are also of potential relevance. Urothelial cells contain a number of xenobiotic-metabolizing enzymes, which enable them to convert pro-carcinogens into reactive intermediates. In a preceding study, it was demonstrated using cultured porcine urinary bladder epithelial cells (PUBEC) that CYP1A1 mRNA is induced in a potent manner by treatment with benzo[a]pyrene (BaP). In the present study, the time dependence of these effects was evaluated and whether PUBEC cultures derived from individual donors respond differently to BaP treatment was determined. CYP1A1 induction was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), and genotoxic effects were studied using the Comet assay. Incubation of PUBEC with BaP increased CYP1A1 expression and induction of DNA strand breaks in a time-dependent manner. Interindividual differences were found between PUBEC cultures derived from several donor animals with respect to the response to BaP, such that the extent of CYP1A1 induction and magnitude of DNA damage was interrelated. Hence, individual differences in metabolic capacities and responsiveness to xenobiotics of urothelial cells from individual donors may be factors in susceptibility to genotoxic effects induced by PAHs.

Structure‐Guided Development of Selective RabGGTase Inhibitors

Designing for selectivity: A combination of protein crystal‐structure analysis, virtual screening, and synthetic chemistry has been used to develop noncytotoxic inhibitors of RabGGTase (IC50: 42 nM for the example shown; red O, blue N, yellow S) that are selective over FTase and GGTase I. Furthermore, the inhibitors display cellular activity and inhibit cancer cell proliferation.

Applicability of superfolder YFP bimolecular fluorescence complementation in vitro.

Abstract Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) is a widely employed method to study protein-protein interactions in cells. As yet, this technique has not been used in vitro. To evaluate a possible application of BiFC in vitro, we constructed a ‘superfolder split YFP’ system where 15 mutations enhance expression of the fusion proteins in Escherichia coli and enable a native purification due to improved solubility. Here, we present the crystal structure of ‘superfolder YFP’, providing the structural basis for the enhanced folding and stability characteristics. Complementation between the two non-fluorescent YFP fragments fused to HRas and Raf1RBD or to 14-3-3 and PMA2-CT52 resulted in the constitution of the functional fluorophore. The in vivo BiFC with these protein interaction pairs was demonstrated in eukaryotic cell lines as well. Here, we present for the first time BiFC in vitro studies with natively purified superfolder YFP fusion proteins and show the potential and drawbacks of this method for analyzing protein-protein interactions.