Identification of histone deacetylase inhibitors with (arylidene)aminoxy scaffold active in uveal melanoma cell lines.

Uveal melanoma (UM) represents an aggressive type of cancer and currently, there is no effective treatment for this metastatic disease. In the last years, histone deacetylase inhibitors (HDACIs) have been studied as a possible therapeutic treatment for UM, alone or in association with other chemotherapeutic agents. Here we synthesised a series of new HDACIs based on the SAHA scaffold bearing an (arylidene)aminoxy moiety. Their HDAC inhibitory activity was evaluated on isolated human HDAC1, 3, 6, and 8 by fluorometric assay and their binding mode in the catalytic site of HDACs was studied by molecular docking. The most promising hit was the quinoline derivative VS13, a nanomolar inhibitor of HDAC6, which exhibited a good antiproliferative effect on UM cell lines at micromolar concentration and a capability to modify the mRNA levels of HDAC target genes similar to that of SAHA.

Design, synthesis, and biological evaluation of stable ß6.3-Helices: Discovery of non-hemolytic antibacterial peptides.

Gramicidin A, a topical antibiotic made from alternating L and D amino acids, is characterized by its wide central pore; upon insertion into membranes, it forms channels that disrupts ion gradients. We present helical peptidomimetics with this characteristic wide central pore that have been designed to mimic gramicidin A channels. Mimetics were designed using molecular modeling focused on oligomers of heterochiral dipeptides of proline analogs, in particular azaproline (AzPro). Molecular Dynamics simulations in water confirmed the stability of the designed helices. A sixteen-residue Formyl-(AzPro-Pro)8-NHCH2CH2OH helix was synthesized as well as a full thirty-two residue Cbz-(AzPro-Pro)16-OtBu channels. No liposomal lysis activity was observed suggesting lack of channel formation, possibly due to inappropriate hydrogen-bonding interactions in the membrane. These peptidomimetics also did not hemolyze red blood cells, unlike gramicidin A.

Limiting Assumptions in the Design of Peptidomimetics.

Preclinical Research Limiting the flexibility of organic compounds to enhance their affinity and selectivity for targeting a macromolecule involved in molecular recognition has become a well-developed paradigm in medicinal chemistry. While the role of reverse-turn motifs as peptidomimetics has received the most attention, ß-sheets and helices are also important motifs for protein/protein interactions. The more complicated problem of mimicking the interacting surface of noncontiguous epitopes will not be considered in this review. This limited overview focuses on efforts to use amino acid synthons as secondary-structure mimetics as well as providing examples of peptidomimetic design focused on nonpeptide synthetic chemistry in contrast. In particular, the rationale of optimal design criteria for mimicry and the many naïve violations of those criteria made in its pursuit are emphasized. Drug Dev Res 78 : 245-267, 2017. © 2017 Wiley Periodicals, Inc.

Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation.

Current antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs), such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators, provides a promising strategy to reduce if not eradicate the viral reservoir. Here, we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly, these isoform-targeted HDAC inhibitors synergize with PKC modulators, namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells.

Structural insights of SmKDAC8 inhibitors: Targeting Schistosoma epigenetics through a combined structure-based 3D QSAR, in vitro and synthesis strategy.

A predictive structure-based 3D QSAR (COMBINEr 2.0) model of the Schistosoma mansoni lysine deacetylase 8 enzyme (SmKDAC8) was developed, validated and used to perform virtual screening (VS) of the NCI Diversity Set V database (1593 compounds). Three external datasets (with congeneric structures to those experimentally resolved in complexes by X-ray and previously reported as SmKDAC8 inhibitors) were employed to compose and validate the most predictive model. Two series characterized by 104 benzodiazepine derivatives (BZDs) and 60 simplified largazole analogs (SLAs), recently reported by our group as human KDAC inhibitors, were tested for their inhibition potency against SmKDAC8 to probe the predictive capability of the quantitative models against compounds with diverse structures. The SmKDAC8 biochemical results confirmed: (1) the benzodiazepine moiety as a valuable scaffold to further investigate when pursuing SmKDAC8 inhibition; (2) the predictive capability of the COMBINEr 2.0 model towards non-congeneric series of compounds, highlighting the most influencing ligand-protein interactions and refining the structure-activity relationships. From the VS investigations, the first 40 top-ranked compounds were obtained and biologically tested for their inhibition potency against SmKDAC8 and hKDACs 1, 3, 6 and 8. Among them, a non-hydroxamic acid benzothiadiazine dioxide derivative (code NSC163639), showed interesting activity and selectivity against SmKDAC8. To further elucidate the structure-activity relationships of NSC163639, two analogs (herein reported as compounds 3 and 4) were synthesized and biologically evaluated. Results suggest the benzothiadiazine dioxide moiety as a promising scaffold to be used in a next step to derive selective SmKDAC8 inhibitors.

Esterase mutation is a mechanism of resistance to antimalarial compounds.

Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance.

Design and synthesis of benzodiazepine analogs as isoform-selective human lysine deacetylase inhibitors.

A comprehensive investigation was performed to identify new benzodiazepine (BZD) derivatives as potent and selective human lysine deacetylase inhibitors (hKDACis). A total of 108 BZD compounds were designed, synthesized and from that 104 compounds were biologically evaluated against human lysine deacetylases (hKDACs) 1, 3 and 8 (class I) and 6 (class IIb). The most active compounds showed mid-nanomolar potencies against hKDACs 1, 3 and 6 and micromolar activity against hKDAC8, while a promising compound (6q) showed selectivity towards hKDAC3 among the different enzyme isoforms. An hKDAC6 homology model, refined by molecular dynamics simulation was generated, and molecular docking studies performed to rationalize the dominant ligand-residue interactions as well as to define structure-activity-relationships. Experimental results confirmed the usefulness of the benzodiazepine moiety as capping group when pursuing hKDAC isoform-selectivity inhibition, suggesting its continued use when designing new hKDACis.

Design and Synthesis of Simplified Largazole Analogues as Isoform-Selective Human Lysine Deacetylase Inhibitors.

Selective inhibition of KDAC isoforms while maintaining potency remains a challenge. Using the largazole macrocyclic depsipeptide structure as a starting point for developing new KDACIs with increased selectivity, a combination of four different simplified largazole analogue (SLA) scaffolds with diverse zinc-binding groups (for a total of 60 compounds) were designed, synthesized, and evaluated against class I KDACs 1, 3, and 8, and class II KDAC6. Experimental evidence as well as molecular docking poses converged to establish the cyclic tetrapeptides (CTPs) as the primary determinant of both potency and selectivity by influencing the correct alignment of the zinc-binding group in the KDAC active site, providing a further basis for developing new KDACIs of higher isoform selectivity and potency.

An Automated Strategy for Binding-Pose Selection and Docking Assessment in Structure-Based Drug Design.

Molecular docking is a widely used technique in drug design to predict the binding pose of a candidate compound in a defined therapeutic target. Numerous docking protocols are available, each characterized by different search methods and scoring functions, thus providing variable predictive capability on a same ligand-protein system. To validate a docking protocol, it is necessary to determine a priori the ability to reproduce the experimental binding pose (i.e., by determining the docking accuracy (DA)) in order to select the most appropriate docking procedure and thus estimate the rate of success in docking novel compounds. As common docking programs use generally different root-mean-square deviation (RMSD) formulas, scoring functions, and format results, it is both difficult and time-consuming to consistently determine and compare their predictive capabilities in order to identify the best protocol to use for the target of interest and to extrapolate the binding poses (i.e., best-docked (BD), best-cluster (BC), and best-fit (BF) poses) when applying a given docking program over thousands/millions of molecules during virtual screening. To reduce this difficulty, two new procedures called Clusterizer and DockAccessor have been developed and implemented for use with some common and "free-for-academics" programs such as AutoDock4, AutoDock4(Zn), AutoDock Vina, DOCK, MpSDockZn, PLANTS, and Surflex-Dock to automatically extrapolate BD, BC, and BF poses as well as to perform consistent cluster and DA analyses. Clusterizer and DockAccessor (code available over the Internet) represent two novel tools to collect computationally determined poses and detect the most predictive docking approach. Herein an application to human lysine deacetylase (hKDAC) inhibitors is illustrated.

Targeting Lysine Deacetylases (KDACs) in Parasites.

Due to an increasing problem of drug resistance among almost all parasites species ranging from protists to worms, there is an urgent need to explore new drug targets and their inhibitors to provide new and effective parasitic therapeutics. In this regard, there is growing interest in exploring known drug leads of human epigenetic enzymes as potential starting points to develop novel treatments for parasitic diseases. This approach of repurposing (starting with validated targets and inhibitors) is quite attractive since it has the potential to reduce the expense of drug development and accelerate the process of developing novel drug candidates for parasite control. Lysine deacetylases (KDACs) are among the most studied epigenetic drug targets of humans, and a broad range of small-molecule inhibitors for these enzymes have been reported. In this work, we identify the KDAC protein families in representative species across important classes of parasites, screen a compound library of 23 hydroxamate- or benzamide-based small molecules KDAC inhibitors, and report their activities against a range of parasitic species, including the pathogen of malaria (Plasmodium falciparum), kinetoplastids (Trypanosoma brucei and Leishmania donovani), and nematodes (Brugia malayi, Dirofilaria immitis and Haemonchus contortus). Compound activity against parasites is compared to that observed against the mammalian cell line (L929 mouse fibroblast) in order to determine potential parasite-versus-host selectivity). The compounds showed nanomolar to sub-nanomolar potency against various parasites, and some selectivity was observed within the small panel of compounds tested. The possible binding modes of the active compounds at the different protein target sites within different species were explored by docking to homology models to help guide the discovery of more selective, parasite-specific inhibitors. This current work supports previous studies that explored the use of KDAC inhibitors in targeting Plasmodium to develop new anti-malarial treatments, and also pioneers experiments with these KDAC inhibitors as potential new anthelminthics. The selectivity observed begins to address the challenges of targeting specific parasitic diseases while limiting host toxicity.

High-resolution crystal structures of protein helices reconciled with three-centered hydrogen bonds and multipole electrostatics.

Theoretical and experimental evidence for non-linear hydrogen bonds in protein helices is ubiquitous. In particular, amide three-centered hydrogen bonds are common features of helices in high-resolution crystal structures of proteins. These high-resolution structures (1.0 to 1.5 Å nominal crystallographic resolution) position backbone atoms without significant bias from modeling constraints and identify Φ = -62°, ψ = -43 as the consensus backbone torsional angles of protein helices. These torsional angles preserve the atomic positions of α-ß carbons of the classic Pauling α-helix while allowing the amide carbonyls to form bifurcated hydrogen bonds as first suggested by Némethy et al. in 1967. Molecular dynamics simulations of a capped 12-residue oligoalanine in water with AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications), a second-generation force field that includes multipole electrostatics and polarizability, reproduces the experimentally observed high-resolution helical conformation and correctly reorients the amide-bond carbonyls into bifurcated hydrogen bonds. This simple modification of backbone torsional angles reconciles experimental and theoretical views to provide a unified view of amide three-centered hydrogen bonds as crucial components of protein helices. The reason why they have been overlooked by structural biologists depends on the small crankshaft-like changes in orientation of the amide bond that allows maintenance of the overall helical parameters (helix pitch (p) and residues per turn (n)). The Pauling 3.6(13) α-helix fits the high-resolution experimental data with the minor exception of the amide-carbonyl electron density, but the previously associated backbone torsional angles (Φ, Ψ) needed slight modification to be reconciled with three-atom centered H-bonds and multipole electrostatics. Thus, a new standard helix, the 3.6(13/10)-, Némethy- or N-helix, is proposed. Due to the use of constraints from monopole force fields and assumed secondary structures used in low-resolution refinement of electron density of proteins, such structures in the PDB often show linear hydrogen bonding.

Helix stability of oligoglycine, oligoalanine, and oligo-ß-alanine dodecamers reflected by hydrogen-bond persistence.

Helices are important structural/recognition elements in proteins and peptides. Stability and conformational differences between helices composed of α- and ß-amino acids as scaffolds for mimicry of helix recognition has become a theme in medicinal chemistry. Furthermore, helices formed by ß-amino acids are experimentally more stable than those formed by α-amino acids. This is paradoxical because the larger sizes of the hydrogen-bonding rings required by the extra methylene groups should lead to entropic destabilization. In this study, molecular dynamics simulations using the second-generation force field, AMOEBA (Ponder, J.W., et al., Current status of the AMOEBA polarizable force field. J Phys Chem B, 2010. 114(8): p. 2549-64.) explored the stability and hydrogen-bonding patterns of capped oligo-ß-alanine, oligoalanine, and oligoglycine dodecamers in water. The MD simulations showed that oligo-ß-alanine has strong acceptor+2 hydrogen bonds, but surprisingly did not contain a large content of 3(12) -helical structures, possibly due to the sparse distribution of the 3(12) -helical structure and other structures with acceptor+2 hydrogen bonds. On the other hand, despite its backbone flexibility, the ß-alanine dodecamer had more stable and persistent <3.0 Å hydrogen bonds. Its structure was dominated more by multicentered hydrogen bonds than either oligoglycine or oligoalanine helices. The 3(1) (PII) helical structure, prevalent in oligoglycine and oligoalanine, does not appear to be stable in oligo-ß-alanine indicating its competition with other structures (stacking structure as indicated by MD analyses). These differences are among the factors that shape helical structural preferences and the relative stabilities of these three oligopeptides.

Limiting assumptions in molecular modeling: electrostatics.

Molecular mechanics attempts to represent intermolecular interactions in terms of classical physics. Initial efforts assumed a point charge located at the atom center and coulombic interactions. It is been recognized over multiple decades that simply representing electrostatics with a charge on each atom failed to reproduce the electrostatic potential surrounding a molecule as estimated by quantum mechanics. Molecular orbitals are not spherically symmetrical, an implicit assumption of monopole electrostatics. This perspective reviews recent evidence that requires use of multipole electrostatics and polarizability in molecular modeling.

Molecular dynamics of ß-hairpin models of epigenetic recognition motifs.

The conformations and stabilities of the ß-hairpin model peptides of Waters (Riemen, A. J.; Waters, M. L. Biochemistry 2009, 48, 1525; Hughes, R. M.; Benshoff, M. L.; Waters, M. L. Chemistry 2007, 13, 5753) have been experimentally characterized as a function of lysine ε-methylation. These models were developed to explore molecular recognition of known epigenetic recognition motifs. This system offered an opportunity to computationally examine the role of cation-π interactions, desolvation of the ε-methylated ammonium groups, and aromatic/aromatic interactions on the observed differences in NMR spectra. AMOEBA, a second-generation force field (Ponder, J. W.; Wu, C.; Ren, P.; Pande, V. S.; Chodera, J. D.; Schnieders, M. J.; Haque, I.; Mobley, D. L.; Lambrecht, D. S.; DiStasio, R. A., Jr.; Head-Gordon, M.; Clark, G. N.; Johnson, M. E.; Head-Gordon, T. J. Phys. Chem. B 2010, 114, 2549), was chosen as it includes both multipole electrostatics and polarizability thought to be essential to accurately characterize such interactions. Independent parametrization of ε-methylated amines was required from which aqueous solvation free energies were estimated and shown to agree with literature values. Molecular dynamics simulations (100 ns) using the derived parameters with model peptides, such as Ac-R-W-V-W-V-N-G-Orn-K(Me)(n)-I-L-Q-NH(2), where n = 0, 1, 2, or 3, were conducted in explicit solvent. Distances between the centers of the indole rings of the two-tryptophan residues, 2 and 4, and the ε-methylated ammonium group on Lys-9 as well as the distance between the N- and C-termini were monitored to estimate the strength and orientation of the cation-π and aromatic/aromatic interactions. In agreement with the experimental data, the stability of the ß-hairpin increased significantly with lysine ε-methylation. The ability of MD simulations to reproduce the observed NOEs for the four peptides was further estimated for the monopole-based force fields, AMBER, CHARMM, and OPLSAA. AMOEBA correctly predicted over 80% of the observed NOEs for all 4 peptides, while the three-monopole force fields were 40-50% predictive in only 2 cases and approximately 10% in the other 10 examples. Preliminary analysis suggests that the decreased cost of desolvation of the substituted ammonium group significantly compensated for the reduced cation-π interaction resulting from the increased separation due to steric bulk of the ε-methylated amines.

Comprehensive model of wild-type and mutant HIV-1 reverse transciptases.

An enhanced version of COMBINE that uses both ligand-based and structure-based alignment of ligands has been used to build a comprehensive 3-D QSAR model of wild-type HIV-1 reverse transcriptase and drug-resistant mutants. The COMBINEr model focused on 7 different RT enzymes complexed with just two HIV-RT inhibitors, niverapine (NVP) and efavirenz (EFV); therefore, 14 inhibitor/enzyme complexes comprised the training set. An external test set of chiral 2-(alkyl/aryl)amino-6-benzylpyrimidin-4(3H)-ones (DABOs) was used to test predictability. The COMBINEr model MC4, although developed using only two inhibitors, predicted the experimental activities of the test set with an acceptable average absolute error of prediction (0.89 pK (i)). Most notably, the model was able to correctly predict the right eudismic ratio for two R/S pairs of DABO derivatives. The enhanced COMBINEr approach was developed using only software freely available to academics.

Histone deacetylase inhibitors: structure-based modeling and isoform-selectivity prediction.

An enhanced version of comparative binding energy (COMBINE) analysis, named COMBINEr, based on both ligand-based and structure-based alignments has been used to build several 3-D QSAR models for the eleven human zinc-based histone deacetylases (HDACs). When faced with an abundance of data from diverse structure-activity sources, choosing the best paradigm for an integrative analysis is difficult. A common example from studies on enzyme-inhibitors is the abundance of crystal structures characterized by diverse ligands complexed with different enzyme isoforms. A novel comprehensive tool for data mining on such inhomogeneous set of structure-activity data was developed based on the original approach of Ortiz, Gago, and Wade, and applied to predict HDAC inhibitors' isoform selectivity. The COMBINEr approach (apart from the AMBER programs) has been developed to use only software freely available to academics.

Inhibition of bacterial virulence: drug-like molecules targeting the Salmonella enterica PhoP response regulator.

Two-component signal transduction (TCST) is the predominant signaling scheme used in bacteria to sense and respond to environmental changes in order to survive and thrive. A typical TCST system consists of a sensor histidine kinase to detect external signals and an effector response regulator to respond to external changes. In the signaling scheme, the histidine kinase phosphorylates and activates the response regulator, which functions as a transcription factor to modulate gene expression. One promising strategy toward antibacterial development is to target TCST regulatory systems, specifically the response regulators to disrupt the expression of genes important for virulence. In Salmonella enterica, the PhoQ/PhoP signal transduction system is used to sense and respond to low magnesium levels and regulates the expression for over 40 genes necessary for growth under these conditions, and more interestingly, genes that are important for virulence. In this study, a hybrid approach coupling computational and experimental methods was applied to identify drug-like compounds to target the PhoP response regulator. A computational approach of structure-based virtual screening combined with a series of biochemical and biophysical assays was used to test the predictability of the computational strategy and to characterize the mode of action of the compounds. Eight compounds from virtual screening inhibit the formation of the PhoP-DNA complex necessary for virulence gene regulation. This investigation served as an initial case study for targeting TCST response regulators to modulate the gene expression of a signal transduction pathway important for bacterial virulence. With the increasing resistance of pathogenic bacteria to current antibiotics, targeting TCST response regulators that control virulence is a viable strategy for the development of antimicrobial therapeutics with novel modes of action.

Limiting assumptions in structure-based design: binding entropy.

In order to deal with the complexity of biological systems at the atomic level, limiting assumptions are often made which do not reflect the reality of the system under study. One example is the assumption that the entropy of binding of the macromolecule is not influenced significantly by the different ligands. Recent experimental data on ligands binding to HIV-1 protease challenge this assumption.

Drug-like leads for steric discrimination between substrate and inhibitors of human acetylcholinesterase.

Protection of the enzyme acetylcholinesterase (AChE) from the toxic effects of organophosphate insecticides and chemical warfare agents (OPs) may be provided by inhibitors that bind at the peripheral binding site (P-site) near the mouth of the active-site gorge. Compounds that bind to this site may selectively block access to the acylation site (A-site) catalytic serine for OPs, but not acetylcholine itself. To identify such compounds, we employed a virtual screening approach using AutoDock 4.2 and AutoDock Vina, confirmed using compounds experimentally known to bind specifically to either the A-site or P-site. Both programs demonstrated the ability to correctly predict the binding site. Virtual screening of the NCI Diversity Set II was conducted using the apo form of the enzyme, and with acetylcholine bound at the crystallographic locations in the A-site only and in both and A- and P-sites. The docking identified 32 compounds that were obtained for testing, and one was demonstrated to bind specifically to the P-site in an inhibitor competition assay.