Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization.

Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features.

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Targeting NOX2 via p47/phox-p22/phox Inhibition with Novel Triproline Mimetics.

On the basis of the knowledge that the proline-rich hot spot PPPRPP region of P(151)PSNPPPRPP(160), an oligopeptide derived from the cytosolic portion of p22phox (p22), binds to the single functional bis-SH3 domain of the regulatory protein p47phox (p47), we designed a mimetic of the tripeptide PPP based on NMR and X-ray crystallographic data for the p22(151-161) peptide PPSNPPPRPPA with a peptide construct. Incorporation of the synthetic pseudo-triproline mimetic Pro-Pro-Cyp in a molecule derived from molecular modeling studies led to only a 7-fold diminution in activity in a surface plasmon resonance assay relative to the same molecule containing the natural Pro-Pro-Pro tripeptide. The alternative sequence corresponding to a Pro-Cyp-Pro insertion was inactive. This is a first example of the use of a triproline mimetic to interfere with the formation of the p47-p22 complex, which is critical for the activation of NOX, leading to the production of reactive oxygen species as superoxide anions.

RNA binding properties of the US11 protein from four primate simplexviruses.

BACKGROUND: The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. METHODS: We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. RESULTS AND CONCLUSIONS: The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.

Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR.

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation.

Role of Fluorination in the Histone Deacetylase 6 (HDAC6) Selectivity of Benzohydroxamate-Based Inhibitors.

Nonselective histone deacetylase (HDAC) inhibitors show dose-limiting side effects due to the inhibition of multiple, essential HDAC subtypes that can be limited or prevented by restricting their selectivity. We herein report the crystal structures of zebrafish HDAC6 catalytic domain 2 (zHDAC6-CD2) in complex with the selective HDAC6 inhibitors ITF3756 and ITF3985 and shed light on the role of fluorination in the selectivity of benzohydroxamate-based structures over class I isoforms. The reason for the enhancement in the selectivity of the benzohydroxamate-based compounds is the presence of specific interactions between the fluorinated linker and the key residues Gly582, Ser531, and His614 of zHDAC6, which are hindered in class I HDAC isoforms by the presence of an Aspartate that replaces Ser531. These results can be used in the design and development of novel, highly selective HDAC6 inhibitors.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

Discovery of an allosteric inhibitor binding site in 3-Oxo-acyl-ACP reductase from Pseudomonas aeruginosa.

3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit-subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH.

Creating a twin STAR.

The Signal Transduction and Activation of RNA (STAR) protein family regulates different levels of RNA metabolism. STAR proteins have been shown to act as homodimers, and the structure of the QUA1 dimerization region of the GLD-1 protein described in this issue (Beuck et al., 2010) provides a link between homodimerization and the selection of RNA targets.

Molecular basis of FIR-mediated c-myc transcriptional control.

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.