Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

Comprehensive Mechanistic View of the Hydrolysis of Oxadiazole-Based Inhibitors by Histone Deacetylase 6 (HDAC6).

Histone deacetylase (HDAC) inhibitors used in the clinic typically contain a hydroxamate zinc-binding group (ZBG). However, more recent work has shown that the use of alternative ZBGs, and, in particular, the heterocyclic oxadiazoles, can confer higher isoenzyme selectivity and more favorable ADMET profiles. Herein, we report on the synthesis and biochemical, crystallographic, and computational characterization of a series of oxadiazole-based inhibitors selectively targeting the HDAC6 isoform. Surprisingly, but in line with a very recent finding reported in the literature, a crystal structure of the HDAC6/inhibitor complex revealed that hydrolysis of the oxadiazole ring transforms the parent oxadiazole into an acylhydrazide through a sequence of two hydrolytic steps. An identical cleavage pattern was also observed both in vitro using the purified HDAC6 enzyme as well as in cellular systems. By employing advanced quantum and molecular mechanics (QM/MM) and QM calculations, we elucidated the mechanistic details of the two hydrolytic steps to obtain a comprehensive mechanistic view of the double hydrolysis of the oxadiazole ring. This was achieved by fully characterizing the reaction coordinate, including identification of the structures of all intermediates and transition states, together with calculations of their respective activation (free) energies. In addition, we ruled out several (intuitively) competing pathways. The computed data (ΔG‡ ≈ 21 kcal·mol-1 for the rate-determining step of the overall dual hydrolysis) are in very good agreement with the experimentally determined rate constants, which a posteriori supports the proposed reaction mechanism. We also clearly (and quantitatively) explain the role of the -CF3 or -CHF2 substituent on the oxadiazole ring, which is a prerequisite for hydrolysis to occur. Overall, our data provide compelling evidence that the oxadiazole warheads can be efficiently transformed within the active sites of target metallohydrolases to afford reaction products possessing distinct selectivity and inhibition profiles.

R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Continuous Assembly of ß-Roll Structures Is Implicated in the Type I-Dependent Secretion of Large Repeat-in-Toxins (RTX) Proteins.

Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight ß strands into blocks that fold into calcium-binding parallel ß-roll structures. The ß-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five ß-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV-V of CyaA (CyaA1372-1681) that consists of a contiguous assembly of two ß-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the "push-ratchet" mechanism of the T1SS-mediated secretion of very large RTX proteins.

Development of PSMA-1007-Related Series of 18F-Labeled Glu-Ureido-Type PSMA Inhibitors.

In recent years, a number of drugs targeting the prostate-specific membrane antigen (PSMA) have become important tools in the diagnosis and treatment of prostate cancer. In the present work, we report on the synthesis and preclinical evaluation of a series of 18F-labeled PSMA ligands for diagnostic application based on the theragnostic ligand PSMA-617. By applying modifications to the linker structure, insight into the structure-activity relationship could be gained, highlighting the importance of hydrophilicity and stereoselectivity on interaction with PSMA and hence the biodistribution. Selected compounds were co-crystallized with the PSMA protein and analyzed by X-rays with mixed results. Among these, PSMA-1007 (compound 5) showed the best interaction with the PSMA protein. The respective radiotracer [18F]PSMA-1007 was translated into the clinic and is, in the meantime, subject of advanced clinical trials.

Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer.

Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

Structural and in Vivo Characterization of Tubastatin A, a Widely Used Histone Deacetylase 6 Inhibitor.

Tubastatin A, a tetrahydro-γ-carboline-capped selective HDAC6 inhibitor (HDAC6i), was rationally designed 10 years ago, and has become the best investigated HDAC6i to date. It shows efficacy in various neurological disease animal models, as HDAC6 plays a crucial regulatory role in axonal transport deficits, protein aggregation, as well as oxidative stress. In this work, we provide new insights into this HDAC6i by investigating the molecular basis of its interactions with HDAC6 through X-ray crystallography, determining its functional capability to elevate the levels of acetylated α-tubulin in vitro and in vivo, correlating PK/PD profiles to determine effective doses in plasma and brain, and finally assessing its therapeutic potential toward psychiatric diseases through use of the SmartCube screening platform.

Novel ß- and γ-Amino Acid-Derived Inhibitors of Prostate-Specific Membrane Antigen.

Prostate-specific membrane antigen (PSMA) is an excellent biomarker for the early diagnosis of prostate cancer progression and metastasis. The most promising PSMA-targeted agents in the clinical phase are based on the Lys-urea-Glu motif, in which Lys and Glu are α-(l)-amino acids. In this study, we aimed to determine the effect of ß- and γ-amino acids in the S1 pocket on the binding affinity for PSMA. We synthesized and evaluated the ß- and γ-amino acid analogues with (S)- or (R)-configuration with keeping α-(l)-Glu as the S1'-binding pharmacophore. The structure-activity relationship studies identified that compound 13c, a ß-amino acid analogue with (R)-configuration, exhibited the most potent PSMA inhibitory activity with an IC50 value of 3.97 nM. The X-ray crystal structure of PSMA in complex with 13c provided a mechanistic basis for the stereochemical preference of PSMA, which can guide the development of future PSMA inhibitors.

Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors.

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific Membrane Antigen Ligands for Potential Use as Theranostics.

The design and synthesis of prostate specific membrane antigen (PSMA) ligands derived from 2-aminoadipic acid, a building block that has not previously been used to construct PSMA ligands, are reported. The effects of both the linker length and of an N-substituent of our PSMA ligands were probed, and X-ray structures of five of these ligands bound to PSMA were obtained. Among the ligands disclosed herein, 13b showed the highest inhibitory activity for PSMA. As ligand 13b can readily be radiolabeled since its fluorine atom is adjacent to the nitrogen atom of its pyridine ring, the use of this and related compounds as theranostics can be pursued.

Calcium-Driven Folding of RTX Domain ß-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into ß-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.