Dissecting Selectivity Determinants of Small-Molecule Inhibitors of SH2 Domains Via Fluorescence Polarization Assays.

Fluorescence polarization (FP) assays can be used to identify small-molecule inhibitors that bind to SH2 domain-containing proteins. We have developed FP assays by which to identify inhibitors of the SH2 domains of the two closely-related transcription factors STAT5a and STAT5b. Point mutation of selected amino acids in the putative binding site of the protein is a valuable tool by which to gain insight into the molecular mechanism of binding. In this chapter, we describe the cloning and application of point mutant proteins in order to transfer the binding preference of selected SH2 domain-binding STAT5b inhibitors to STAT5a, with results that highlight the importance of considering a role for residues outside the SH2 domain in contributing to the binding interactions of SH2 domain inhibitors.

A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4.

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z'-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.

Effect of Amino Acid Substitutions on 70S Ribosomal Binding, Cellular Uptake, and Antimicrobial Activity of Oncocin Onc112.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high-priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram-negative and Gram-positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram-negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off-target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

Prevalence of Capsular Serotype, Pilus Island Distribution, and Antibiotic Resistance in Pediatric and Adult Invasive Group B Streptococcus Isolates: Data From a Nationwide Prospective Surveillance Study in Germany.

For neonates, group B Streptococcus is life threatening. Current prevention strategies remain insufficient, especially for cases of late-onset sepsis, where intrapartum antibiotic prophylaxis has demonstrated no benefit. One promising approach is the vaccination of pregnant women, which offers protective immunity via transplacental transmission of neutralizing antibodies. Our nationwide, prospective surveillance study aimed to characterize the prevalence of pilus antigen, capsular polysaccharide serotypes, and antibiotic resistance from invasive GBS infections in neonates and compare these results with those from children and adults in Germany. Our study includes 173 neonatal isolates of a total of 450 reported cases during the study period (incidence: 0.34/1000 live births), in addition to 2 pediatric and 803 adult isolates. The comparison between neonatal and adult isolates reveals age-dependent differences in capsular serotype and pilus type distribution and differences in antibiotic resistance patterns.

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate.

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

ATP Inhibits the Transcription Factor STAT5b.

Although naturally occurring low-molecular-weight compounds have many known roles within the cell, these do not usually involve the direct inhibition of protein-protein interactions. Based on the results of high-throughput screening of a library of bioactive compounds and neurotransmitters, we report here that the four nucleoside triphosphates ATP, GTP, CTP and UTP inhibit the SH2 domain of the tumor-related transcription factor STAT5b. ATP and GTP are the most active nucleoside triphosphates and show specificity for STAT5b over STAT5a, STAT3, STAT6 and the p53-binding protein HDM2. As the inhibition constant of ATP against STAT5b is significantly lower than published values for the intracellular ATP concentration, our data suggest that ATP might inhibit the protein-protein interactions of STAT5b in living cells.

The STAT5b Linker Domain Mediates the Selectivity of Catechol Bisphosphates for STAT5b over STAT5a.

STAT family proteins are important mediators of cell signaling and represent therapeutic targets for the treatment of human diseases. Most STAT inhibitors target the protein-protein interaction domain, the SH2 domain, but specificity for a single STAT protein is often limited. Recently, we developed catechol bisphosphates as the first inhibitors of STAT5b demonstrated to exhibit a high degree of selectivity over the close homologue STAT5a. Here, we show that the amino acid in position 566 of the linker domain, not the SH2 domain, is the main determinant of specificity. Arg566 in wild-type STAT5b favors tight binding of catechol bisphosphates, while Trp566 in wild-type STAT5a does not. Amino acid 566 also determines the affinity for a tyrosine-phosphorylated peptide derived from the EPO receptor for STAT5a and STAT5b, demonstrating the functional relevance of the STAT5 linker domain for the adjacent SH2 domain. These results provide the first demonstration that a residue in the linker domain can determine the affinity of nonpeptidic small-molecule inhibitors for the SH2 domain of STAT proteins. We propose targeting the interface between the SH2 domain and linker domain as a novel design approach for the development of potent and selective STAT inhibitors. In addition, our data suggest that the linker domain could contribute to the enigmatically divergent biological functions of the two STAT5 proteins.

A selective inhibitor of the Polo-box domain of Polo-like kinase 1 identified by virtual screening.

Polo-like kinase 1 (PLK1), a member of the Polo-like kinase family, plays an important regulatory role in mitosis and cell cycle progression. PLK1 overexpression is correlated with tumourigenesis and poor prognosis in cancer patients. Therefore, the identification of novel compounds that inhibit PLK1 would provide attractive therapeutic approaches. Although some PLK1 kinase inhibitors have been developed, their application has been limited by off-target effects. PLK1 contains a regulatory domain named the Polo-box domain (PBD), which is characteristic only for the Polo-like kinase family. This domain represents an alternative therapeutic target with higher selectivity for PLK1. In this study, we applied in silico virtual drug screening, fluorescence polarization and microscale thermophoresis to identify new scaffolds targeting the PBD of PLK1. One compound, 3-{[(1R,9S)-3-(naphthalen-2-yl)-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-11-yl]methyl}benzonitrile (designated compound (1)), out of a total of 30,793 natural product derivatives, inhibited the PLK1 PBD with high selectivity (IC50: 17.9 ±â€¯0.5 µM). This compound inhibited the growth of cultured leukaemia cells (CCRF-CEM and CEM/ADR5000) and arrested the cell cycle in the G2/M phase, which is characteristic for PLK1 inhibitors. Immunofluorescence analyses showed that treatment with compound (1) disrupted spindle formation due to the aberrant localization of PLK1 during the mitotic process, leading to G2/M arrest and ultimately cell death. In conclusion, compound (1) is a selective PLK1 inhibitor that inhibits cancer cell growth. It represents a chemical scaffold for the future synthesis of new selective PLK1 inhibitors for cancer therapy.

Development of Erasin: a chromone-based STAT3 inhibitor which induces apoptosis in Erlotinib-resistant lung cancer cells.

Inhibition of protein-protein interactions by small molecules offers tremendous opportunities for basic research and drug development. One of the fundamental challenges of this research field is the broad lack of available lead structures from nature. Here, we demonstrate that modifications of a chromone-based inhibitor of the Src homology 2 (SH2) domain of the transcription factor STAT5 confer inhibitory activity against STAT3. The binding mode of the most potent STAT3 inhibitor Erasin was analyzed by the investigation of structure-activity relationships, which was facilitated by chemical synthesis and biochemical activity analysis, in combination with molecular docking studies. Erasin inhibits tyrosine phosphorylation of STAT3 with selectivity over STAT5 and STAT1 in cell-based assays, and increases the apoptotic rate of cultured NSCLC cells in a STAT3-dependent manner. This ability of Erasin also extends to HCC-827 cells with acquired resistance against Erlotinib, a clinically used inhibitor of the EGF receptor. Our work validates chromone-based acylhydrazones as privileged structures for antagonizing STAT SH2 domains, and demonstrates that apoptosis can be induced in NSCLC cells with acquired Erlotinib resistance by direct inhibition of STAT3.

A small-molecule screen identifies the antitrypanosomal agent suramin and analogues NF023 and NF449 as inhibitors of STAT5a/b.

The transcription factor STAT5a is a potential target for tumor therapy. We present a fluorescence polarization-based, high-throughput screen of chemical libraries containing natural products and known bioactive molecules, for the identification of small-molecule inhibitors of the STAT5a SH2 domain. This screen identified suramin, a drug used to treat African trypanosomiasis, and its analogues NF023 and NF449 as inhibitors of the SH2 domains of STAT5a/b. Our data extend the known in vitro targets of suramin and analogues to include members of the STAT protein family.

Rational development of Stafib-2: a selective, nanomolar inhibitor of the transcription factor STAT5b.

The transcription factor STAT5b is a target for tumour therapy. We recently reported catechol bisphosphate and derivatives such as Stafib-1 as the first selective inhibitors of the STAT5b SH2 domain. Here, we demonstrate STAT5b binding of catechol bisphosphate by solid-state nuclear magnetic resonance, and report on rational optimization of Stafib-1 (Ki = 44 nM) to Stafib-2 (Ki = 9 nM). The binding site of Stafib-2 was validated using combined isothermal titration calorimetry (ITC) and protein point mutant analysis, representing the first time that functional comparison of wild-type versus mutant protein by ITC has been used to characterize the binding site of a small-molecule ligand of a STAT protein with amino acid resolution. The prodrug Pomstafib-2 selectively inhibits tyrosine phosphorylation of STAT5b in human leukaemia cells and induces apoptosis in a STAT5-dependent manner. We propose Pomstafib-2, which currently represents the most active, selective inhibitor of STAT5b activation available, as a chemical tool for addressing the fundamental question of which roles the different STAT5 proteins play in various cell processes.

Evaluation of a Dry EEG System for Application of Passive Brain-Computer Interfaces in Autonomous Driving.

We tested the applicability and signal quality of a 16 channel dry electroencephalography (EEG) system in a laboratory environment and in a car under controlled, realistic conditions. The aim of our investigation was an estimation how well a passive Brain-Computer Interface (pBCI) can work in an autonomous driving scenario. The evaluation considered speed and accuracy of self-applicability by an untrained person, quality of recorded EEG data, shifts of electrode positions on the head after driving-related movements, usability, and complexity of the system as such and wearing comfort over time. An experiment was conducted inside and outside of a stationary vehicle with running engine, air-conditioning, and muted radio. Signal quality was sufficient for standard EEG analysis in the time and frequency domain as well as for the use in pBCIs. While the influence of vehicle-induced interferences to data quality was insignificant, driving-related movements led to strong shifts in electrode positions. In general, the EEG system used allowed for a fast self-applicability of cap and electrodes. The assessed usability of the system was still acceptable while the wearing comfort decreased strongly over time due to friction and pressure to the head. From these results we conclude that the evaluated system should provide the essential requirements for an application in an autonomous driving context. Nevertheless, further refinement is suggested to reduce shifts of the system due to body movements and increase the headset's usability and wearing comfort.

Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1.

Polo-like kinase 1 (Plk1), a key player in mitosis, is overexpressed in a wide range of tumor types and has been validated as a target for tumor therapy. In addition to its N-terminal kinase domain, Plk1 harbors a C-terminal protein-protein interaction domain, referred to as the polo-box domain (PBD). Because the PBD is unique to the five-member family of polo-like kinases, and its inhibition is sufficient to inhibit the enzyme, the Plk1 PBD is an attractive target for the inhibition of Plk1 function. Although peptide-based inhibitors are invaluable tools for elucidating the nature of the binding interface, small molecules are better suited for the induction of mitotic arrest and apoptosis in tumor cells by Plk1 inhibition. This review describes the considerable progress that has been made in developing small-molecule and peptide-based inhibitors of the Plk1 PBD.

Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent and Selective Inhibitors of the Transcription Factor STAT5b.

Design approaches for inhibitors of protein-protein interactions are rare, but highly sought after. Here, we report that O-phosphorylation of simple derivatives of the natural products dihydrocapsaicin and N-vanillylnonanamide leads to inhibitors of the SH2 domain of the transcription factor STAT5b. The most potent molecule is obtained from dihydrocapsaicin in only three synthetic steps. It has submicromolar affinity for the SH2 domain of STAT5b (Ki = 0.34 µM), while displaying 35-fold selectivity over the highly homologous STAT5a (Ki = 13.0 µM). The corresponding pivaloyloxymethyl ester inhibits STAT5b with selectivity over STAT5a in human tumor cells. Importantly, it inhibits cell viability and induces apoptosis in human tumor cells in a STAT5-dependent manner. Our data validate O-phosphorylation of appropriately preselected natural products or natural product derivatives as a semirational design approach for small molecules that selectively inhibit phosphorylation-dependent protein-protein interaction domains in cultured human tumor cells.

Nanomolar inhibitors of the transcription factor STAT5b with high selectivity over STAT5a.

Src homology 2 (SH2) domains play a central role in signal transduction. Although many SH2 domains have been validated as drug targets, their structural similarity makes development of specific inhibitors difficult. The cancer-relevant transcription factors STAT5a and STAT5b are particularly challenging small-molecule targets because their SH2 domains are 93% identical on the amino acid level. Here we present the natural product-inspired development of the low-nanomolar inhibitor Stafib-1, as the first small molecule which inhibits the STAT5b SH2 domain (K(i)=44 nM) with more than 50-fold selectivity over STAT5a. The binding site of the core moiety of Stafib-1 was validated by functional analysis of point mutants. A prodrug of Stafib-1 was shown to inhibit STAT5b with high selectivity over STAT5a in tumor cells. Stafib-1 provides the first demonstration that naturally occurring SH2 domains with more than 90% sequence identity can be selectively targeted with small organic molecules.

Wing and body kinematics of takeoff and landing flight in the pigeon (Columba livia).

Takeoff and landing are critical phases in a flight. To better understand the functional importance of the kinematic adjustments birds use to execute these flight modes, we studied the wing and body movements of pigeons (Columba livia) during short-distance free-flights between two perches. The greatest accelerations were observed during the second wingbeat of takeoff. The wings were responsible for the majority of acceleration during takeoff and landing, with the legs contributing only one-quarter of the acceleration. Parameters relating to aerodynamic power output such as downstroke amplitude, wingbeat frequency and downstroke velocity were all greatest during takeoff flight and decreased with each successive takeoff wingbeat. This pattern indicates that downstroke velocity must be greater for accelerating flight to increase the amount of air accelerated by the wings. Pigeons used multiple mechanisms to adjust thrust and drag to accelerate during takeoff and decelerate during landing. Body angle, tail angle and wing plane angles all shifted from more horizontal orientations during takeoff to near-vertical orientations during landing, thereby reducing drag during takeoff and increasing drag during landing. The stroke plane was tilted steeply downward throughout takeoff (increasing from -60+/-5 deg. to -47+/-1 deg.), supporting our hypothesis that a downward-tilted stroke plane pushes more air rearward to accelerate the bird forward. Similarly, the stroke plane tilted upward during landing (increasing from -1+/-2 deg. to 17+/-7 deg.), implying that an upward-tilted stroke plane pushes more air forward to slow the bird down. Rotations of the stroke plane, wing planes and tail were all strongly correlated with rotation of the body angle, suggesting that pigeons are able to redirect aerodynamic force and shift between flight modes through modulation of body angle alone.

Kinematics and power requirements of ascending and descending flight in the pigeon (Columba livia).

Ascending or descending locomotion involves a change in potential energy (PE) and a corresponding change in power requirement. We sought to test whether the mechanical power required for steady ascending or descending flight is a simple sum of the power required for level flight and the power necessary for potential energy change. Pigeons (Columba livia) were trained to fly at varying angles of ascent and descent (60 degrees , 30 degrees , 0 degrees , -30 degrees , -60 degrees ), and were recorded using high-speed video. Detailed three-dimensional kinematics were obtained from the recordings, allowing analysis of wing movement. Aerodynamic forces and power requirements were then estimated from kinematic data. As expected, ;PE flight power' increased significantly with angle of flight (0.234 W deg.(-1)), though there appeared to be a limit on the amount of PE that the birds could gain or dissipate per wingbeat. We found that the total power output for flight at various angles was not different from the sum of power required for level flight and the PE rate of change for a given angle, except for the steep -60 degrees descent. The total power for steep descent was higher than this sum because of a higher induced power due to the bird's deceleration and slower flight velocity. Aerodynamic force estimates during mid-downstroke did not differ significantly in magnitude or orientation among flight angles. Pigeons flew fastest during -30 degrees flights (4.9+/-0.1 m s(-1)) and slowest at 60 degrees (2.9+/-0.1 m s(-1)). Although wingbeat frequency ranged from 6.1 to 9.6 Hz across trials, the variation was not significant across flight angles. Stroke plane angle was more horizontal, and the wing more protracted, for both +60 degrees and -60 degrees flights, compared with other flight path angles.