Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density.

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Discovery of sulfonamides and 9-oxo-2,8-diazaspiro[5,5]undecane-2-carboxamides as human kynurenine aminotransferase 2 (KAT2) inhibitors.

Human kynurenine aminotransferase 2 (KAT2) inhibitors could be potentially used to treat the cognitive deficits associated with bipolar disease and schizophrenia. Although, there has been active drug research activity by several industrial and academic groups in developing KAT2 inhibitors over the years, no such compound has proceeded to the clinics. Here, we report two different chemical series of reversible KAT2 inhibitors with sub-micromolar activities. The first series was identified by a high-throughput screening of a diverse random library and the second one by structure-based virtual screening. Two novel crystal structures of KAT2 complexed with different reversible inhibitors were also deposited to the Protein databank which could be useful for future drug discovery efforts.

Discovery of novel Cyclophilin D inhibitors starting from three dimensional fragments with millimolar potencies.

Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.

Design and Evaluation of Highly Selective Human Immunoproteasome Inhibitors Reveal a Compensatory Process That Preserves Immune Cell Viability.

The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic cells. Here, we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome cocrystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.

Total Synthesis of Covalent Cysteine Protease Inhibitor N-Desmethyl Thalassospiramide C and Crystallographic Evidence for Its Mode of Action.

A total synthesis of N-desmethyl thalassospiramide C, a unique strained macrocyclic proteobacterial depsipeptide, enabled a detailed crystallographic study of its covalent complex with cathepsin K, a member of a medicinally important family of cysteine proteases. The study provides support for the mechanism of action, and the insight gained can be used for structure-based drug design targeting these calpain proteases.

Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator.

New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation.IMPORTANCEPseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.

Crystal structure of the catalytic domain of human atypical protein kinase C-iota reveals interaction mode of phosphorylation site in turn motif.

Atypical protein kinases C (aPKCs) play critical roles in signaling pathways that control cell growth, differentiation and survival. Therefore, they constitute attractive targets for the development of novel therapeutics against cancer. The crystal structure of the catalytic domain of atypical PKCiota in complex with the bis(indolyl)maleimide inhibitor BIM1 has been determined at 3.0A resolution within the frame of the European Structural Proteomics Project SPINE. The overall structure exhibits the classical bilobal kinase fold and is in its fully activated form. Both phosphorylation sites (Thr403 in the activation loop, and Thr555 in the turn motif) are well defined in the structure and form intramolecular ionic contacts that make an important contribution in stabilizing the active conformation of the catalytic subunit. The phosphorylation site in the hydrophobic motif of atypical PKCs is replaced by the phosphorylation mimic glutamate and this is also clearly seen in the structure of PKCiota (residue 574). This structure determination for the first time provides the architecture of the turn motif phosphorylation site, which is characteristic for PKCs and PKB/AKT, and is completely different from that in PKA. The bound BIM1 inhibitor blocks the ATP-binding site and puts the kinase domain into an intermediate open conformation. The PKCiota-BIM1 complex is the first kinase domain crystal structure of any atypical PKC and constitutes the basis for rational drug design for selective PKCiota inhibitors.

Structure of MrsD, an FAD-binding protein of the HFCD family.

MrsD from Bacillus sp. HIL-Y85/54728 is a member of the HFCD (homo-oligomeric flavin-containing Cys decarboxylases) family of flavoproteins and is involved in the biosynthesis of the lantibiotic mersacidin. It catalyses the oxidative decarboxylation of the C-terminal cysteine residue of the MrsA precursor peptide of mersacidin, yielding a (Z)-enethiol intermediate as the first step in the formation of the unusual amino acid S-[(Z)-2-aminovinyl]-methyl-D-cysteine. Surprisingly, MrsD was found to bind FAD, in contrast to the three other characterized members of the HFCD family, which bind FMN. To determine the molecular discriminators of FAD binding within the HFCD family, the crystal structure of MrsD was analyzed at a resolution of 2.54 A. Crystals of space group F432 contain one MrsD monomer in the asymmetric unit. However, a Patterson search with EpiD-derived models failed. Based on the consideration that the dodecameric MrsD particle of tetrahedral symmetry resembles the quaternary structure of EpiD, rotational and translational parameters were derived from the geometric consideration that the MrsD dodecamer is generated from a monomer by crystallographic symmetry around the position (1/4, 1/4, 1/4) of the unit cell. A structural comparison with the FMN-binding members of the HFCD family EpiD and AtHAL3a shows conserved sequence motifs in contact with the flavin's pyrimidine ring but divergent environments for the dimethylbenzene ring of the isoalloxazine moiety. The position of the ribityl chain differs in MrsD from that found in EpiD and AtHAL3a. However, the FMN-phosphate binding sites are also highly conserved in their exact positions. In all three cases, the flavin cofactor is bound to a structurally conserved region of the Rossmann-fold monomer, exposing its Re side for catalysis. The adenosyl phosphate of FAD is anchored in a well defined binding site and the adenosine moieties are oriented towards the interior of the hollow particle, where three of them pack against each other around the threefold axis of a trimeric facet.

Crystal structure of the plant PPC decarboxylase AtHAL3a complexed with an ene-thiol reaction intermediate.

The Arabidopsis thaliana protein AtHAL3a decarboxylates 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, a step in coenzyme A biosynthesis. Surprisingly, this decarboxylation reaction is carried out as an FMN-dependent redox reaction. In the first half-reaction, the side-chain of the cysteine residue of 4'-phosphopantothenoylcysteine is oxidised and the thioaldehyde intermediate decarboxylates spontaneously to the 4'-phosphopantothenoyl-aminoethenethiol intermediate. In the second half-reaction this compound is reduced to 4'-phosphopantetheine and the FMNH(2) cofactor is re-oxidised. The active site mutant C175S is unable to perform this reductive half-reaction. Here, we present the crystal structure of the AtHAL3a mutant C175S in complex with the reaction intermediate pantothenoyl-aminoethenethiol and FMNH(2). The geometry of binding suggests that reduction of the C(alpha)=C(beta) double bond of the intermediate can be performed by direct hydride-transfer from N5 of FMNH(2) to C(beta) of the aminoethenethiol-moiety supported by a protonation of C(alpha) by Cys175. The binding mode of the substrate is very similar to that previously observed for a pentapeptide to the homologous enzyme EpiD that introduces the aminoethenethiol-moiety as final reaction product at the C terminus of peptidyl-cysteine residues. This finding further supports our view that these homologous enzymes form a protein family of homo-oligomeric flavin-containing cysteine decarboxylases, which we have termed HFCD family.