Crystal Structure of a Histone Deacetylase Homologue from <i>Pseudomonas aeruginosa</i>

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 <i>Pseudomonas aeruginosa</i> 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

Disparate SAR Data of Griseofulvin Analogues for the Dermatophytes Trichophyton mentagrophytes, T. rubrum, and MDA-MB-231 Cancer Cells

Griseofulvin and 53 analogues of this compound have been tested against the pathogenic dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes as well as against the breast cancer cell line MDA-MB-231. The modifications to griseofulvin include the 4, 5, 6, 2′, 3′, and 4′ positions. The SAR of the griseofulvin analogues toward the two fungi followed the same trend with the majority being less active than griseofulvin and none had more than twice the potency of the parent compound. A comparison of the antifungal and the anticancer SAR revealed distinct differences, as the majority of analogues showed increased activity against the cancer cell line MDA-MB-231, highlighted by 2′-benzyloxy-2′-demethoxy-griseofulvin, which showed low activity against both fungi but was among the most potent compounds against MDA-MB-231 cancer cells. Tubulin has been proposed as the target of griseofulvin in both fungal and mammalian cells, but the differences revealed by this SAR study strongly suggest that the mode-of-action of the compound class toward fungi and mammalian cancer cells is different

A Functional [NiFe]-Hydrogenase Model Compound That Undergoes Biologically Relevant Reversible Thiolate Protonation

Two model compounds of the active site of [NiFe]-hydrogenases with an unusual {S₂Ni­(μ-S)­(μ-CO)­Fe­(CO)₂S}-coordination environment around the metals are reported. The neutral compound [Ni­(xbsms)­(μ-CO)­(μ-S)­Fe­(CO)₂(‘S’)], (<b>1</b>) (H₂xbsms = 1,2-bis­(4-mercapto-3,3-dimethyl-2-thiabutyl)­benzene) is converted to [<b>1</b>H]­[BF₄] by reversible protonation using HBF₄·Et₂O. The protonation takes place at the terminal thiolate sulfur atom that is coordinated to nickel. Catalytic intermediates with a protonated terminal cysteinate were suggested for the native protein but have not yet been confirmed experimentally. [<b>1</b>H]­[BF₄] is the first dinuclear [NiFe] model compound for such a species. Both complexes have been synthesized and characterized by X-ray crystallography, NMR-, FTIR-, and ⁵⁷Fe-Mössbauer spectroscopy as well as by electronic absorption and resonance Raman spectroscopy. The experimental results clearly show that the protonation has a significant impact on the electronic structure of the iron center, although it takes place at the nickel site. DFT calculations support the interpretation of the spectroscopic data and indicate the presence of a bonding interaction between the metal ions, which is relevant for the enzyme as well. Electrochemical experiments show that both <b>1</b> and [<b>1</b>H]­[BF₄] are active for electrocatalytic proton reduction in aprotic solvents

A Functional [NiFe]-Hydrogenase Model Compound That Undergoes Biologically Relevant Reversible Thiolate Protonation

Two model compounds of the active site of [NiFe]-hydrogenases with an unusual {S₂Ni­(μ-S)­(μ-CO)­Fe­(CO)₂S}-coordination environment around the metals are reported. The neutral compound [Ni­(xbsms)­(μ-CO)­(μ-S)­Fe­(CO)₂(‘S’)], (<b>1</b>) (H₂xbsms = 1,2-bis­(4-mercapto-3,3-dimethyl-2-thiabutyl)­benzene) is converted to [<b>1</b>H]­[BF₄] by reversible protonation using HBF₄·Et₂O. The protonation takes place at the terminal thiolate sulfur atom that is coordinated to nickel. Catalytic intermediates with a protonated terminal cysteinate were suggested for the native protein but have not yet been confirmed experimentally. [<b>1</b>H]­[BF₄] is the first dinuclear [NiFe] model compound for such a species. Both complexes have been synthesized and characterized by X-ray crystallography, NMR-, FTIR-, and ⁵⁷Fe-Mössbauer spectroscopy as well as by electronic absorption and resonance Raman spectroscopy. The experimental results clearly show that the protonation has a significant impact on the electronic structure of the iron center, although it takes place at the nickel site. DFT calculations support the interpretation of the spectroscopic data and indicate the presence of a bonding interaction between the metal ions, which is relevant for the enzyme as well. Electrochemical experiments show that both <b>1</b> and [<b>1</b>H]­[BF₄] are active for electrocatalytic proton reduction in aprotic solvents

A Functional [NiFe]-Hydrogenase Model Compound That Undergoes Biologically Relevant Reversible Thiolate Protonation

Two model compounds of the active site of [NiFe]-hydrogenases with an unusual {S₂Ni­(μ-S)­(μ-CO)­Fe­(CO)₂S}-coordination environment around the metals are reported. The neutral compound [Ni­(xbsms)­(μ-CO)­(μ-S)­Fe­(CO)₂(‘S’)], (<b>1</b>) (H₂xbsms = 1,2-bis­(4-mercapto-3,3-dimethyl-2-thiabutyl)­benzene) is converted to [<b>1</b>H]­[BF₄] by reversible protonation using HBF₄·Et₂O. The protonation takes place at the terminal thiolate sulfur atom that is coordinated to nickel. Catalytic intermediates with a protonated terminal cysteinate were suggested for the native protein but have not yet been confirmed experimentally. [<b>1</b>H]­[BF₄] is the first dinuclear [NiFe] model compound for such a species. Both complexes have been synthesized and characterized by X-ray crystallography, NMR-, FTIR-, and ⁵⁷Fe-Mössbauer spectroscopy as well as by electronic absorption and resonance Raman spectroscopy. The experimental results clearly show that the protonation has a significant impact on the electronic structure of the iron center, although it takes place at the nickel site. DFT calculations support the interpretation of the spectroscopic data and indicate the presence of a bonding interaction between the metal ions, which is relevant for the enzyme as well. Electrochemical experiments show that both <b>1</b> and [<b>1</b>H]­[BF₄] are active for electrocatalytic proton reduction in aprotic solvents

How Formaldehyde Inhibits Hydrogen Evolution by [FeFe]-Hydrogenases: Determination by ¹³C ENDOR of Direct Fe–C Coordination and Order of Electron and Proton Transfers

Formaldehyde (HCHO), a strong electrophile and a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases, is used to identify the point in the catalytic cycle at which a highly reactive metal-hydrido species is formed. Investigations of the reaction of Chlamydomonas reinhardtii [FeFe]-hydrogenase with formaldehyde using pulsed-EPR techniques including electron–nuclear double resonance spectroscopy establish that formaldehyde binds close to the active site. Density functional theory calculations support an inhibited super-reduced state having a short Fe–¹³C bond in the 2Fe subsite. The adduct forms when HCHO is available to compete with H⁺ transfer to a vacant, nucleophilic Fe site: had H⁺ transfer already occurred, the reaction of HCHO with the Fe-hydrido species would lead to methanol, release of which is not detected. Instead, Fe-bound formaldehyde is a metal-hydrido mimic, a locked, inhibited form analogous to that in which two electrons and only one proton have transferred to the H-cluster. The results provide strong support for a mechanism in which the fastest pathway for H₂ evolution involves two consecutive proton transfer steps to the H-cluster following transfer of a second electron to the active site

Inhibition of [FeFe]-Hydrogenases by Formaldehyde and Wider Mechanistic Implications for Biohydrogen Activation

Formaldehydea rapid and reversible inhibitor of hydrogen evolution by [FeFe]-hydrogenasesbinds with a strong potential dependence that is almost complementary to that of CO. Whereas exogenous CO binds tightly to the oxidized state known as H_ox but very weakly to a state two electrons more reduced, formaldehyde interacts most strongly with the latter. Formaldehyde thus intercepts increasingly reduced states of the catalytic cycle, and density functional theory calculations support the proposal that it reacts with the H-cluster directly, most likely targeting an otherwise elusive and highly reactive Fe-hydrido (Fe–H) intermediate

Radiosynthesis and Preclinical Evaluation of 3′-Aza-2′‑[¹⁸F]fluorofolic Acid: A Novel PET Radiotracer for Folate Receptor Targeting

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [¹⁸F]­fluoride (integrated approach). 3′-Azafolic acid and its 2′-halogenated derivatives (2′-chloro and 2′-fluoro) were evaluated in vitro to determine their binding affinity. 3′-Aza-2′-[¹⁸F]­fluorofolic acid ([¹⁸F]<b>6</b>) was obtained, starting from <i>N</i>²-acetyl-3′-aza-2′-chlorofolic acid di-<i>tert</i>-butylester (<b>2</b>), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35–127 GBq/μmol. Binding affinity to the FR was high (IC₅₀ = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 °C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 °C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [¹⁸F]<b>6</b> in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new ¹⁸F-labeled 3′-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue

Macrocyclization of Quinazoline-Based EGFR Inhibitors Leads to Exclusive Mutant Selectivity for EGFR L858R and Del19

Activating mutations in the epidermal growth factor receptor (EGFR) are frequent oncogenic drivers of non-small-cell lung cancer (NSCLC). The most frequent alterations in EGFR are short in-frame deletions in exon 19 (Del19) and the missense mutation L858R, which both lead to increased activity and sensitization of NSCLC to EGFR inhibition. The first approved EGFR inhibitors used for first-line treatment of NSCLC, gefitinib and erlotinib, are quinazoline-based. However, both inhibitors have several known off-targets, and they also potently inhibit wild-type (WT) EGFR, resulting in side effects. Here, we applied a macrocyclic strategy on a quinazoline-based scaffold as a proof-of-concept study with the goal of increasing kinome-wide selectivity of this privileged inhibitor scaffold. Kinome-wide screens and SAR studies yielded 3f, a potent inhibitor for the most common EGFR mutation (EGFR Del19: 119 nM) with selectivity against the WT receptor (EGFR: >10 μM) and the kinome