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Human histone deacetylase 4 (HDAC4) belongs to class IIa of the zinc-dependent histone deacetylases. HDAC4 is an established target for various indication areas, in particular Huntington's disease, heart failure and cancer. To reduce unwanted side effects, it is advantageous to develop isozyme-selective inhibitors, which poses a major challenge due to the highly conserved active centers of the HDAC family. According to current knowledge it is assumed that H976 in HDAC4 wt occurs exclusively in the out-conformation and thus the selective foot pocket is constitutively open. In contrast, the side chain of the corresponding tyrosine in HDAC4 H976Y adopts the in-conformation, and is thus able to stabilize the intermediate state of the deacetylation reaction and block access to the foot pocket. In this study, we provide evidence that a dynamic equilibrium exists between the in- and out-conformation in HDAC4 wt . The binding of selective HDAC4 inhibitors that address the foot pocket can be enhanced in HDAC4 variants with mainly small, but also medium hydrophobic or polar side chains. We attribute this to the fact that these side chains are preferentially present in the out-conformation. Therefore, we propose HDAC4 H976A and other HDAC4 variants as promising tools to find and enrich HDAC4-selective foot pocket binders in screening campaigns that might have been overlooked in conventional screens with HDAC4 wt ., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Franz-Josef Meyer-Almes reports financial support was provided by Government of the State of Hessen. Frederik Lermyte reports financial support was provided by German Research Foundation. Frederik Lermyte reports financial support was provided by Government of the State of Hessen. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone deacetylase 4 (HDAC4) contributes to gene repression by complex formation with HDAC3 and the corepressor silencing mediator for retinoid or thyroid hormone receptors (SMRT). We hypothesized that peptides derived from the class IIa specific binding site of SMRT would stabilize a specific conformation of its target protein and modulate its activity. Based on the SMRT-motif 1 (SM1) involved in the interaction of SMRT with HDAC4, we systematically developed cyclic peptides that exhibit K i values that are 9 to 56 times lower than that of the linear SMRT peptide. The peptide macrocycles stabilize the wildtype of the catalytic domain of HDAC4 (cHDAC4) considerably better than its thermally more stable 'gain-of-function' (GOF) variant, cHDAC4-H976Y. Molecular docking and mutagenesis studies indicated that the cyclic peptides bind in a similar but not identical manner as the linear SMRT peptide to a discontinuous binding site. Ion mobility mass spectrometry showed no major changes in the protein fold upon peptide binding. Consistent with these results, preliminary hydrogen-deuterium exchange mass spectrometry measurements indicated only minor conformational changes. Taken together, the cyclic SMRT peptides most likely stabilize the apo form of cHDAC4., (© 2024 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The kinetics and mechanism of drug binding to its target are critical to pharmacological efficacy. A high throughput (HTS) screen often results in hundreds of hits, of which usually only simple IC 50 values are determined during reconfirmation. However, kinetic parameters such as residence time for reversible inhibitors and the k inact / K I ratio, which is the critical measure for evaluating covalent inactivators, are early predictive measures to assess the chances of success of the hits in the clinic. Using the promising cancer target human histone deacetylase 8 as an example, we present a robust method that calculates concentration-dependent apparent rate constants for the inhibition or inactivation of HDAC8 from dose-response curves recorded after different pre-incubation times. With these data, hit compounds can be classified according to their mechanism of action, and the relevant kinetic parameters can be calculated in a highly parallel fashion. HDAC8 inhibitors with known modes of action were correctly assigned to their mechanism, and the binding mechanisms of some hits from an internal HDAC8 screening campaign were newly determined. The oxonitriles SVE04 and SVE27 were classified as fast reversible HDAC8 inhibitors with moderate time-constant IC 50 values of 4.2 and 2.6 µM, respectively. The hit compound TJ-19-24 and SAH03 behave like slow two-step inactivators or reversible inhibitors, with a very low reverse isomerization rate.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn 2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Five-membered heteroaromatic rings, in particular, have gained prominence in medicinal chemistry as they offer enhanced metabolic stability, solubility and bioavailability, crucial factors in developing effective drugs. The unique physicochemical properties and biological effects of five-membered heterocycles have positioned them as key structural motifs in numerous clinically effective drugs. Hence, the exploration of five-ring heterocycles remains an important research area in medicinal chemistry, with the aim of discovering new therapeutic agents for various diseases. This review addresses the incorporation of heteroatoms such as nitrogen, oxygen and sulfur into the aromatic ring of these heterocyclic compounds, enhancing their polarity and facilitating both aromatic stacking interactions and the formation of hydrogen bonds. Histone deacetylases are present in numerous multiprotein complexes within the epigenetic machinery and play a central role in various cellular processes. They have emerged as important targets for cancer, neurodegenerative diseases and other therapeutic indications. In histone deacetylase inhibitors (HDACi's), five-ring heterocycles perform various functions as a zinc-binding group, a linker or head group, contributing to binding activity and selective recognition. This review focuses on providing an up-to-date overview of the different five-membered heterocycles utilized in HDACi motifs, highlighting their biological properties. It summarizes relevant publications from the past decade, offering insights into the recent advancements in this field of research.

A series of novel quinazoline-4-(3H)-one derivatives were designed and synthesized as histone deacetylase 6 (HDAC6) inhibitors based on novel quinazoline-4-(3H)-one as the cap group and benzhydroxamic acid as the linker and metal-binding group. A total of 19 novel quinazoline-4-(3H)-one analogues ( 5a - 5s ) were obtained. The structures of the target compounds were characterized using 1 H-NMR, 13 C-NMR, LC-MS, and elemental analyses. Characterized compounds were screened for inhibition against HDAC8 class I, HDAC4 class IIa, and HDAC6 class IIb. Among the compounds tested, 5b proved to be the most potent and selective inhibitor of HDAC6 with an IC 50 value 150 nM. Some of these compounds showed potent antiproliferative activity in several tumor cell lines (HCT116, MCF7, and B16). Amongst all the compounds tested for their anticancer effect against cancer cell lines, 5c emerged to be most active against the MCF-7 line with an IC 50 of 13.7 μM; it exhibited cell-cycle arrest in the G2 phase, as well as promoted apoptosis. Additionally, we noted a significant reduction in the colony-forming capability of cancer cells in the presence of 5c . At the intracellular level, selective inhibition of HDAC6 was enumerated by monitoring the acetylation of α-tubulin with a limited effect on acetyl-H3. Importantly, the obtained results suggested a potent effect of 5c at sub-micromolar concentrations as compared to the other molecules as HDAC6 inhibitors in vitro.

Aim: Human histone deacetylase 8 (KDAC8) is a well-recognized pharmaceutical target in Cornelia de Lange syndrome and different types of cancer, particularly childhood neuroblastoma. Several classes of chemotypes have been identified, which interfere with the enzyme activity of KDAC8. These compounds have been identified under equilibrium or near equilibrium conditions for inhibitor binding to the target enzyme. This study aims for the classification of KDAC8 inhibitors according to the mode of action and identification of most promising lead compounds for drug development., Methods: A continuous enzyme activity assay is used to monitor inhibition kinetics., Results: A high-throughput continuous KDAC8 activity assay is developed that provides additional mechanistic information about enzyme inhibition enabling the classification of KDAC8 inhibitors according to their mode of action. Fast reversible inhibitors act as a molecular chaperone and are capable to rescue the enzyme activity of misfolded KDAC8, while covalent inactivators and slow dissociating inhibitors do not preserve KDAC8 activity., Conclusions: The application of continuous KDAC8 activity assay reveals additional information about the mode of interaction with inhibitors, which can be used to classify KDAC8 inhibitors according to their mode of action. The approach is compatible with the high-throughput screening of compound libraries. Fast reversible inhibitors of KDAC8 act as molecular chaperones and recover enzyme activity from misfolded protein conformations. In contrast, slow-binding inhibitors and covalent inactivators of KDAC8 are not capable to recover enzyme activity., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2023.)

Cyanoacrylates define a class of inhibitors which are capable to form a transient covalent bond with a cysteine flanking the binding site, thereby increasing the residence time and prolonging the inhibitory effect on the target protein under nonequilibrium conditions. Herein, we describe the synthetic access to cyanoacrylate-based HDAC4 inhibitors and the procedures for the characterization of the transient nature of the covalent bond between cyanoacrylates and thiols or cysteines in HDAC4., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

HDAC8 is an important target in several indication areas including childhood neuroblastoma. Several isozyme selective inhibitors of HDAC8 with L-shaped structures have been developed. A theoretical study has suggested that methionine 274 (M274) would act as a "switch" that controls a transient binding pocket, which is induced upon binding of L-shaped inhibitors. This hypothesis was experimentally examined in this study. The thermostability and functionality of HDAC8 wildtype and mutant variants with exchanged M274 were analyzed using biophysical methods. Furthermore, the binding kinetics of L-shaped and linear reference inhibitors of these HDAC8 variants were determined in order to elucidate the mode of interaction. Exchange of M274 has considerable impact on enzyme activity, but is not the decisive factor for selective recognition of HDAC8 by L-shaped inhibitors.

Histone deacetylases (HDACs) play an essential role in the transcriptional regulation of cells through the deacetylation of nuclear histone and non-histone proteins and are promising therapeutic targets for the treatment of various diseases. Here, the synthesis of new compounds in which a hydroxamic acid residue is attached to differently substituted pyrimidine rings via a methylene group bridge of varying length as potential HDAC inhibitors is described. The target compounds were obtained by alkylation of 2-(alkylthio)pyrimidin-4(3 H )-ones with ethyl 2-bromoethanoate, ethyl 4-bromobutanoate, or methyl 6-bromohexanoate followed by aminolysis of the obtained esters with hydroxylamine. Oxidation of the 2-methylthio group to the methylsulfonyl group and following treatment with amines resulted in the formation of the corresponding 2-amino-substituted derivatives, the ester group of which reacted with hydroxylamine to give the corresponding hydroxamic acids. The synthesized hydroxamic acids were tested as inhibitors of the HDAC4 and HDAC8 isoforms. Among the synthesized pyrimidine-based hydroxamic acids N -hydroxy-6-[6-methyl-2-(methylthio)-5-propylpyrimidin-4-yloxy]hexanamide was found to be the most potent inhibitor of both the HDAC4 and HDAC8 isoforms, with an IC 50 of 16.6 µM and 1.2 µM, respectively., (Copyright © 2022, Jakubkiene et al.)

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds 13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments. It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.) also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Angiogenesis deregulation is often linked to cancer and is thus an essential target. Materials & methods: Twenty-nine compounds were developed as VEGFR-2 inhibitors. Compounds were evaluated to determine their antiangiogenic activity. Results: B1, PB11 and PB16 showed HUVEC's IC 50  scores in the submicromolar range. B1, B2 and PB16 reduced cellular migration and capillary tube formation of HUVECs. VEGFR-2 inhibitory activity was found in the nanomolar range: 200 nM of B1, 500 nM of B2 and 600 nM of PB16. B1 and PB16 suppressed the formation of new capillaries on growing CAMs. B1 and PB16 occupied the ATP site and allosteric pocket of VEGFR-2 in docking studies. Conclusion: These compounds can target VEGFR-2 and are endowed with in vitro and in vivo antiangiogenic activity.

Recently, we have reported that non-hydroxamate thiazolidinedione (TZD) analogs are capable of inhibiting human deacetylase 4 (HDAC4). This study aims at the dissection of the molecular determinants and kinetics of the molecular recognition of TZD ligands by HDAC4. For this purpose, a structure activity relationship analysis of 225 analogs was combined with a comprehensive study of the enzyme and binding kinetics of a variety of HDAC4 mutant variants. The experimental data were rationalized by docking to the two major conformations of HDAC4. TZD ligands are competitive inhibitors and bind via a two-step mechanism involving principal molecular recognition and induced fit. The residence time of 24 g is (34 ± 3) min and thus much larger than that of the canonical pan-HDAC inhibitor SAHA ((5 ± 2) min). Importantly, the binding kinetics can be tuned by varying the structure of the CAP group.

Histone deacetylases (HDACs) remove acetyl groups from acetylated lysine residues and have a large variety of substrates and interaction partners. Therefore, it is not surprising that HDACs are involved in many diseases. Most inhibitors of zinc-dependent HDACs (HDACis) including approved drugs contain a hydroxamate as a zinc-binding group (ZBG), which is by far the biggest contributor to affinity, while chemical variation of the residual molecule is exploited to create more or less selectivity against HDAC isozymes or other metalloproteins. Hydroxamates have a propensity for nonspecificity and have recently come under considerable suspicion because of potential mutagenicity. Therefore, there are significant concerns when applying hydroxamate-containing compounds as therapeutics in chronic diseases beyond oncology due to unwanted toxic side effects. In the last years, several alternative ZBGs have been developed, which can replace the critical hydroxamate group in HDACis, while preserving high potency. Moreover, these compounds can be developed into highly selective inhibitors. This review aims at providing an overview of the progress in the field of non-hydroxamic HDACis in the time period from 2015 to present. Formally, ZBGs are clustered according to their binding mode and structural similarity to provide qualitative assessments and predictions based on available structural information.

In anticancer drug discovery, multi-targeting compounds have been beneficial due to their advantages over single-targeting compounds. For instance, VEGFR-2 has a crucial role in angiogenesis and cancer management, whereas HDACs are well-known regulators of epigenetics and have been known to contribute significantly to angiogenesis and carcinogenesis. Herein, we have reported nineteen novel VEGFR-2 and HDAC dual-targeting analogs containing diaryl-pyrazoline thiazolidinediones and their in vitro and in vivo biological evaluation. In particular, the most promising compound 14c has emerged as a dual inhibitor of VEGFR-2 and HDAC. It demonstrated anti-angiogenic activity by inhibiting in vitro HUVEC proliferation, migration, and tube formation. Moreover, an in vivo CAM assay showed that 14c repressed new capillary formation in CAMs. In particular, 14c exhibited cytotoxicity potential on different cancer cell lines such as MCF-7, K562, A549, and HT-29. Additionally, 14c demonstrated significant potency and selectivity against HDAC4 in the sub-micromolar range. To materialize the hypothesis, we also performed molecular docking on the crystal structures of both VEGFR-2 (PDB ID: 1YWN) and HDAC4 (PDB-ID: 4CBY), which corroborated the designing and biological activity. The results indicated that compound 14c could be a potential lead to develop more optimized multi-target analogs with enhanced potency and selectivity., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Monotargeting anticancer agents suffer from resistance and target nonspecificity concerns, which can be tackled with a multitargeting approach. The combined treatment with HDAC inhibitors and PPARγ agonists has displayed potential antitumor effects. Based on these observations, this work involves design and synthesis of molecules that can simultaneously target PPARγ and HDAC. Several out of 25 compounds inhibited HDAC4, and six compounds acted as dual-targeting agents. Compound 7i was the most potent, with activity toward PPARγ EC 50 = 0.245 μM and HDAC4 IC 50 = 1.1 μM. Additionally, compounds 7c and 7i were cytotoxic to CCRF-CEM cells (CC 50 = 2.8 and 9.6 μM, respectively), induced apoptosis, and caused DNA fragmentation. Furthermore, compound 7c modulated the expression of c-Myc, cleaved caspase-3, and caused in vivo tumor regression in CCRF-CEM tumor xenografts. Thus, this study provides a basis for the rational design of dual/multitargeting agents that could be developed further as anticancer therapeutics.

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes., (© 2020 Wiley-VCH GmbH.)

In search for new and safer anti-cancer agents, a structurally guided pharmacophore hybridization strategy of two privileged scaffolds, namely diaryl pyrazolines and imidazolidine-2,4-dione (hydantoin), was adopted resulting in a newfangled series of compounds (H1-H22). Herein, a bio-isosteric replacement of "pyrrolidine-2,5-dione" moiety of our recently reported antitumor hybrid incorporating diaryl pyrazoline and pyrrolidine-2,5-dione scaffolds with "imidazoline-2,4-dione" moiety has been incorporated. Complete biological studies revealed the most potent analog among all i.e. compound H13, which was at-least 10-fold more potent compared to the corresponding pyrrolidine-2,5-dione, in colon and breast cancer cells. In-vitro studies showed activation of caspases, arrest of G0/G1 phase of cell cycle, decrease in the expression of anti-apoptotic protein (Bcl-2) and increased DNA damage. In-vivo assay on HT-29 (human colorectal adenocarcinoma) animal xenograft model unveiled the significant anti-tumor efficacy along with oral bioavailability with maximum TGI 36% (i.p.) and 44% (per os) at 50 mg/kg dose. These findings confirm the suitability of hybridized pyrazoline and imidazolidine-2,4-dione analog H13 for its anti-cancer potential and starting-point for the development of more efficacious analogs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cancer is a heterogeneous disease, and its treatment requires the identification of new ways to thwart tumor cells. Amongst such emerging targets are glucose transporters (GLUTs, SLC2 family), which are overexpressed by almost all types of cancer cells; their inhibition provides a strategy to disrupt tumor metabolism selectively, leading to antitumor effects. Here, novel thiazolidinedione (TZD) derivatives were designed, synthesized, characterized, and evaluated for their GLUT1, GLUT4, and GLUT5 inhibitory potential, followed by in-vitro cytotoxicity determination in leukemic cell lines. Compounds G5, G16, and G17 inhibited GLUT1, with IC 50 values of 5.4 ± 1.3, 26.6 ± 1.8, and 12.6 ± 1.2 μM, respectively. G17 was specific for GLUT1, G16 inhibited GLUT4 (IC 50  = 21.6 ± 4.5 μM) comparably but did not affect GLUT5. The most active compound, G5, inhibited all three GLUT types, with GLUT4 IC 50  = 9.5 ± 2.8 μM, and GLUT5 IC 50  = 34.5 ± 2.4 μM. Docking G5, G16, and G17 to the inward- and outward-facing structural models of GLUT1 predicted ligand binding affinities consistent with the kinetic inhibition data and implicated E380 and W388 of GLUT1 vs. their substitutions in GLUT5 (A388 and A396, respectively) in inhibitor preference for GLUT1. G5 inhibited the proliferation of leukemia CEM cells at low micromolar range (IC 50  = 13.4 μM) while being safer for normal blood cells. Investigation of CEM cell cycle progression after treatment with G5 showed that cells accumulated in the G2/M phase. Flow cytometric apoptosis studies revealed that compound G5 induced both early and late-stage apoptosis in CEM cells., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human histone deacetylase 8 is a well-recognized target for T-cell lymphoma and particularly childhood neuroblastoma. PD-404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys 102 and Cys 153 . This study on the distinct effects of PD-404,182 on HDAC8 reveals that this compound induces the dose-dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD-404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC 50 =0.78±0.01 μM), HT29 (IC 50 =0.92±0.15 μM) and K562 (IC 50 =47.25±1.24 μM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G 1 /G 0 phase and decreased cell population in G 2 /M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg -1 dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents., (© 2020 Wiley-VCH GmbH.)

3CL proteases (3CL pro ) are only found in RNA viruses and have a central role in polyprotein processing during replication. Therefore, 3CL pro has emerged as promising drug target for therapeutic treatment of infections caused by Coronaviruses. In the light of the recent major outbreak of the SARS-CoV-2 virus and the continuously rising numbers of infections and casualties, there is an urgent need for quickly available drugs or vaccines to stop the current COVID-19 pandemic. Repurposing of approved drugs as 3CL pro inhibitors could dramatically shorten the period up to approval as therapeutic against SARS-CoV-2, since pharmacokinetics and toxicity is already known. Several known drugs, e.g. oxytetracycline, doxorubicin, kanamycin, cefpiramide, teniposide, proanthocyanidin and salvianolic acid B, but also not-approved active compounds from the ZINC15 library were identified as new potential inhibitors of 3CL pro by using different complementary virtual screening and docking approaches. These compounds have the potential to be further optimized using structure based drug design as demonstrated for oxytetracycline., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC 50 11.4 and 14.7 μM, respectively). F18 was equally selective for GLUT4 (IC 50 6.8 μM), while F19 was specific for GLUT1 (IC 50 152 μM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC 50 of 1.7 μM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC 50 - 9.3 μM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC 50 - 28.2 μM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC 50 - 104.2 μM) and human fibroblasts (HS27) (CC 50 - 105.0 μM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in various indication areas, including different types of cancer and particularly childhood neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was found to be most potent selective inhibitors of HDAC8 with IC 50 values of 2.7 μM and 6.3 μM respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3 h both were found to induce apoptosis and cause cell cycle arrest in G2/M phase., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human histone deacetylase 8 (HDAC8) is a highly promising target for neuroblastoma and other types of cancer. Several HDAC inhibitors are approved for the treatment of special cancer subtypes or are evaluated in clinical trials. By far the most drugs or drug candidates contain a hydroxamate group that chelates the catalytic zinc ion within HDACs. Most hydroxamate inhibitors are more or less unselective, although there are considerable exceptions demonstrating the general feasibility to develop at least HDAC isoenzyme selective inhibitors. In addition, hydroxamates have recently come under discussion regarding their potential for mutagenicity. Recently, PD-404,182 was discovered as a selective and potent non-hydroxamate inhibitor of HDAC8. However, this active compound turned out to be decomposed in the presence of glutathion (GSH). Here, we describe the synthesis of significantly improved analogs of PD-404,182 that demonstrate both, great selectivity for HDAC8 and also chemical stability in the presence of GSH. The compounds are characterized with respect to structure-activity relationship, binding mode and target engagement in neuroblastoma cells by combining biochemical and biophysical methods with chemoinformatics., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

An efficient and practical thiocarbonyl surrogate via combination of potassium sulfide and chloroform was established. A variety of dithiocarbamates were afforded along with four new chemical bond formations in a one-pot reaction in which the thiocarbonyl motif was generated in situ. Furthermore, these readily accessed molecules showed promising activity against HDAC8, opening a potential gateway to discover a new type of nonhydroxamate and isoenzyme-selective HDAC inhibitors.

This article places its focus on methods and tools enabling the elucidation of the mechanism by which ligands, small-molecule inhibitors, or substrates interact with zinc-containing bacterial or human members of the histone deacetylase family (HDACs). These methods include biochemical and biophysical approaches and can be subdivided into equilibrium and kinetic methods. More information about the exact mode of action can be obtained by combining these methods with specific mutant variants of the enzymes and/or series of structural similar ligands. All available equilibrium and kinetic data including additional information from 3D structures of HDAC-ligand complexes can be beneficially combined in a data analysis procedure called Integrated Global-Fit analysis eventually providing the most likely binding mechanism., (© 2018 John Wiley & Sons A/S.)

Histone deacetylase 8 (HDAC8) is an established and validated target for T-cell lymphoma and childhood neuroblastoma. The active site binding pocket of HDAC8 is highly conserved among all zinc-containing representatives of the histone deacetylase (HDAC) family. This explains that most HDACs are unselectively recognized by similar inhibitors featuring a zinc binding group (ZBG), a hydrophobic linker and a head group. In the light of this difficulty, the creation of isoenzyme-selectivity is one of the major challenges in the development of HDAC inhibitors. In a series of trifluoromethylketone inhibitors of HDAC8 compound 10 shows a distinct binding mechanism and a dramatically increased residence time (RT) providing kinetic selectivity against HDAC4. Combining the binding kinetics results with computational docking and binding site flexibility analysis suggests that 10 occupies the conserved catalytic site as well as an adjacent transient sub-pocket of HDAC8.

Background: HDAC8 is an established target for T-cell lymphoma and childhood neuroblastoma. Benzothiazine-imines are promising HDAC8 inhibitors with unknown binding mechanism lacking a usual zinc binding group., Methods: In this study high-resolution and quantitative HPLC-coupled ESI-MS/MS techniques are combined with crystal structure determination and a variety of biochemical and computational methods to elucidate the reaction mechanism between benzothiazine-imine 1 and HDAC8., Results: 1) 1 is a covalent inhibitor of HDAC8; 2) inhibition is reversible in the presence of reducing agents; 3) C153 in the active site and C102 are involved in the inhibition mechanism; 4) 1 modifies various cysteines in HDAC8 forming either thiocyanates or mixed disulfides with 3; 5) 1 and 5 dock in close proximity to C153 within the active site. This is supposed to accelerate covalent inactivation particularly in HDAC8 and suggested as major determinant for the observed nanomolar potency and selectivity of 1., Conclusions: 1 and its analogs are interesting model compounds but unsuitable for therapeutic treatment due to their high unselective reactivity towards thiol groups. However, the postulated preceding non-covalent binding mode of 1 opens a door to optimized next generation compounds that combine potent and selective non-covalent recognition with low reactivity towards C153 at the active site of HDAC8., General Significance: 1 represents a completely new class of inhibitors for HDAC8. Initial non-covalent interaction at the bottom of the active site is suggested to be the key for its selectivity. Further optimization of non-covalent interaction and thiol-reactivity provides opportunities to develop therapeutic useful covalent HDAC8 inhibitors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enzymes from the histone deacetylase (HDAC) family are highly regulated by different mechanisms. However, only very limited knowledge exists about the regulation of HDAC8, an established target in multiple types of cancer. A previous dedicated study of HDAC class I enzymes identified no redox-sensitive cysteinyl thiol in HDAC8. This is in contrast to the observation that HDAC8 preparations show different enzyme activities depending on the addition of reducing agents. In the light of the importance of HDAC8 in tumorigenesis a possible regulation by redox signaling was investigated using biochemical and biophysical methods combined with site directed mutagenesis. The occurrence of a characteristic disulfide bond under oxidizing conditions is associated with a complete but reversible loss of enzyme activity. Cysteines 102 and 153 are the integral components of the redox-switch. A possible regulation of HDAC8 by redox signal transduction is suggested by the observed relationship between inhibition of reactive oxygen species generating NOX and concomitant increased HDAC8 activity in neuroblastoma tumor cells. The slow kinetics for direct oxidation of HDAC8 by hydrogen peroxide suggests that transmitters of oxidative equivalents are required to transfer the H 2 O 2 signal to HDAC8., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Industrialized drug screening campaigns usually deliver hundreds of compounds that are active on a particular pharmaceutical target. In light of high failure rates of drug candidates due to unforeseeable off-target toxicity, the early identification of the most promising compounds with high potential for target selectivity is an urgent need to improve the quality of lead compounds and lower attrition rates in the drug development process. The reliable prediction of the selectivity of active substances for a target protein is a challenging task. A comprehensive study of the binding kinetics, thermodynamics, and selectivity of chemically related ligands of histone deacetylase (HDAC) like amidohydrolase from Pseudomonas aeruginosa (HDAH pa ) reveals one general binding mechanism for all analyzed compounds consisting of a preceding conformational selection step followed by an optional subsequent induced fit. Depending on the chemical structure, the ligands bind to one or two of at least three protein conformations with different rate constants. Although these kinetic and mechanistic differences hamper the predictability of selectivity for the HDAC inhibitors, we demonstrate that the enthalpy-weighted binding constant KdΔH is a useful metric to predict isoform selectivity of inhibitors against HDAC enzymes and relatively robust toward different but related binding mechanisms., (© 2017 John Wiley & Sons A/S.)

Fluorescence lifetime (FLT) is a robust intrinsic property and material constant of fluorescent matter. Measuring this important physical indicator has evolved from a laboratory curiosity to a powerful and established technique for a variety of applications in drug discovery, medical diagnostics and basic biological research. This distinct trend was mainly driven by improved and meanwhile affordable laser and detection instrumentation on the one hand, and the development of suitable FLT probes and biological assays on the other. In this process two essential working approaches emerged. The first one is primarily focused on high throughput applications employing biochemical in vitro assays with no requirement for high spatial resolution. The second even more dynamic trend is the significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging. The latter approach is currently pursued to enable not only the investigation of immortal tumor cell lines, but also specific tissues or even organs in living animals. This review tries to give an actual overview about the current status of FLT based bioassays and the wide range of application opportunities in biomedical and life science areas. In addition, future trends of FLT technologies will be discussed.

Background: The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied., Methods: Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases., Results: The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation., Conclusions: The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes., General Significance: The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A series of perfluorinated SAHA (PFSAHA) was prepared and profiled against a panel of human and bacterial members of the Histone deacetylase (HDAC) family. Some of the active substances show nanomolar inhibitory activity and several hundred fold selectivity for the HDAC like enzyme PA3774 from P. aeruginosa. The extraordinary selectivity against human HDACs results from the distinct oligomeric state of PA3774 which consists of two head-to-head dimers. The binding pocket is defined by the surface of both opposite monomers confining the access of ligands to the active site. In addition, the aromatic cap group of PFSAHA undergoes an edge-to-face aromatic interaction with phenylalanine from the opposite monomer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Class IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as "Readers" of acetyl marks, whereas enzymatically active HDACs of class I or IIb are called "Erasers" to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of class IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to class I HDAC8., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC 50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z-isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

Despite the recently growing interest in the acetylation of lysine residues by prokaryotic enzymes, the underlying biological function is still not well understood. Deacetylation is accomplished by proteins that belong to the histone deacetylase (HDAC) superfamily. In this report, we present the first crystal structure of PA3774, a histone deacetylase homologue from the human pathogen Pseudomonas aeruginosa that shares a high degree of homology with class IIb HDACs. We determined the crystal structure of the ligand-free enzyme and protein-ligand complexes with a trifluoromethylketone inhibitor and the reaction product acetate. Moreover, we produced loss of function mutants and determined the structure of the inhibitor-free PA3774 H143A mutant, the inhibitor-free PA3774 Y313F mutant, and the PA3774 Y313F mutant in complex with the highly selective hydroxamate inhibitor PFSAHA. The overall structure reveals that the exceptionally long L1 loop mediates the formation of a tetramer composed of two "head-to-head" dimers. The distinctive dimer interface significantly confines the entrance area of the active site, suggesting a crucial role for substrate recognition and selectivity.

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T-cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2-c][1,3]benzothiazin-6-imine scaffold was studied that is completely different from the traditional concept of HDAC inhibitors comprising a zinc binding group (ZBG), in most cases a hydroxamate group, a spacer, and a capping group that may interact with the surface of the target protein. Although lacking a ZBG, some of the new compounds were shown to have outstanding potency against HDAC8 in the single-digit nanomolar range. The pyrimido[1,2-c][1,3]benzothiazin-6-imines also inhibited the growth of solid and hematological tumor cells. The small size and beneficial physicochemical properties of the novel HDAC inhibitor class underline the high degree of drug likeness. This and the broad structure-activity relationship suggest great potential for the further development of compounds with the pyrimido[1,2-c][1,3]benzothiazin-6-imine scaffold into innovative and highly effective therapeutic drugs against cancer., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular recognition between proteins and small molecule ligands is at the heart of biological function in cellular systems and the basis of modern rational drug development. Therefore, the mechanisms governing protein-ligand interaction have been objects of research for many decades. The last 15 years has seen a revival of a discussion whether conformational selection (CS) or induced fit (IF) is the most relevant binding mechanism. A decreasing observed rate constant, k obs, with increasing ligand concentration was considered to be a hallmark of CS, but according to contemporary knowledge, a positive saturating behavior of k obs can be explained by both CS and IF mechanisms. The only currently recognized kinetic method to differentiate between both binding mechanisms includes the measurement of two separate series of binding kinetics with variation of either protein or ligand under pseudo-first-order conditions. This study avoids the disadvantage of high protein concentrations and provides evidence that a comprehensive Integrated Global Fit analysis of sets of binding kinetics with just varied ligand concentration in combination with equilibrium data and optional displacement kinetics can effectively differentiate between CS and IF binding mechanisms. The limiting situation, when physical binding dominates over the previous (CS) or subsequent (IF) conformational changes, is carefully analyzed. Finally, the relevance of kinetic methods and the elucidation of more complex binding mechanisms are discussed for advanced rational selection and optimization of drug candidates.

Background: Pseudomonas aeruginosa, a Gram-negative, aerobic coccobacillus bacterium is an opportunistic human pathogen and worldwide the fourth most common cause of hospital-acquired infections which are often high mortality such as ventilator-associated pneumoniae. The polyamine metabolism of P. aeruginosa and particularly the deacetylation of acetylpolyamines has been little studied up to now. Results with other bacterial pathogens e.g., Y. pestis suggest that polyamines may be involved in the formation of biofilms or confer resistance against certain antibiotics., Results: To elucidate the role of acetylpolyamines and their enzymatic deacetylation in more detail, all three putative acetylpolyamine amidohydrolases (APAHs) from P. aeruginosa have been expressed in enzymatic active form. The APAHs PA0321 and PA1409 are shown to be true polyamine deacetylases, whereas PA3774 is not able to deacetylate acetylated polyamines. Every APAH can hydrolyze trifluoroacetylated lysine-derivatives, but only PA1409 and much more efficiently PA3774 can also process the plain acetylated lysine substrate. P. aeruginosa is able to utilize acetylcadaverine and acetylputrescine as a carbon source under glucose starvation. If either the PA0321 or the PA1409 but not the PA3774 gene is disrupted, the growth of P. aeruginosa is reduced and delayed. In addition, we were able to show that the APAH inhibitors SAHA and SATFMK induce biofilm formation in both PA14 and PAO1 wildtype strains., Conclusions: P. aeruginosa has two functional APAHs, PA0321 and PA1409 which enable the utilization of acetylpolyamines for the metabolism of P. aeruginosa. In contrast, the physiological role of the predicted APAH, PA3774, remains to be elucidated. Its ability to deacetylate synthetic acetylated lysine substrates points to a protein deacetylation functionality with yet unknown substrates.

The importance of binding kinetics in terms of residence time and on-rate in drug discovery has been broadly accepted in the past few years. Furthermore, evidence has accumulated that the optimal binding mechanism of a drug to its target molecule is related to physiological efficacy as well as selectivity and thus drug safety. Homogeneous fluorescence-based binding assays have been shown to enable high throughput kinetics requiring only small amounts of protein and can be developed to elucidate even complex mechanisms of molecular recognition. A generalized approach is proposed that combines high quality kinetic and equilibrium data in an Integrated Global Fit analysis yielding the most probable binding mechanism., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The photocontrol of protein function like enzyme activity has been the subject of many investigations to enable reversible and spatiotemporally defined cascading biochemical reactions without the need for separation in miniaturized and parallelized assay setups for academic and industrial applications. A photoswitchable amidohydrolase variant from Bordetella/Alcaligenes with the longest reported half-life (approximately 30 h) for the cis-state of the attached azobenzene group was chosen as a model system to dissect the underlying mechanism and molecular interactions that caused the enormous deceleration of the thermal cis-to-trans relaxation of the azobenzene photoswitch. A systematic site-directed mutagenesis study on the basis of molecular dynamics simulation data was employed to investigate enzyme and thermal cis-to-trans relaxation kinetics in dependence on selected amino acid substitution, which revealed a prominent histidine and a hydrophobic cluster as molecular determinants for the stabilization of the cis-isomer of the attached azobenzene moiety on the protein surface. The nature of the involved interactions consists of polar, hydrophobic, and possibly aromatic Π-Π contributions. The elucidated principles behind the stabilization of the cis-state of azobenzene derivatives on a protein surface can be exploited to design improved biologically inspired photoswitches. Moreover, the findings open the door to highly long-lived cis-states of azobenzene groups yielding improved bistable photoswitches that can be controlled by single light-pulses rather than continuous irradiation with UV light that causes potential photodamage to the employed biomolecules., (Copyright © 2015 John Wiley & Sons, Ltd.)

A series of azobenzenealkylmaleimides (AMDs) with different spacer length was synthesized and coupled via Michael-Addition to a specific mutant of a bacterial histone deacetylase-like amidohydrolase (HDAH). Michaelis-Menten parameters (Vmax and Km) were employed to characterize the effect of both, the spacer length and the configuration (cis vs. trans) of the attached azobenzene moiety, on the HDAH enzyme activity. The photoswitch behavior of the AMD/enzyme conjugate activity was clearly influenced by the AMD spacer length. This study highlights the importance of steric rearrangement of the photoswitch with respect to the active site and describes a strategy to optimize the photocontrol of HDAH., (Copyright © 2014 Elsevier Inc. All rights reserved.)