Selectivity of Hydroxamate- and Difluoromethyloxadiazole-Based Inhibitors of Histone Deacetylase 6 In Vitro and in Cells.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

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

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

Optimization of Drug Candidates That Inhibit the D-Loop Activity of RAD51.

RAD51 is the central protein in homologous recombination (HR) repair, where it first binds ssDNA and then catalyzes strand invasion via a D-loop intermediate. Additionally, RAD51 plays a role in faithful DNA replication by protecting stalled replication forks; this requires RAD51 to bind DNA but may not require the strand invasion activity of RAD51. We previously described a small-molecule inhibitor of RAD51 named RI(dl)-2 (RAD51 inhibitor of D-loop formation #2, hereafter called 2 h), which inhibits D-loop activity while sparing ssDNA binding. However, 2 h is limited in its ability to inhibit HR in vivo, preventing only about 50 % of total HR events in cells. We sought to improve upon this by performing a structure-activity relationship (SAR) campaign for more potent analogues of 2 h. Most compounds were prepared from 1-(2-aminophenyl)pyrroles by forming the quinoxaline moiety either by condensation with aldehydes, then dehydrogenation of the resulting 4,5-dihydro intermediates, or by condensation with N,N'-carbonyldiimidazole, chlorination, and installation of the 4-substituent through Suzuki-Miyaura coupling. Many analogues exhibited enhanced activity against human RAD51, but in several of these compounds the increased inhibition was due to the introduction of dsDNA intercalation activity. We developed a sensitive assay to measure dsDNA intercalation, and identified two analogues of 2 h that promote complete HR inhibition in cells while exerting minimal intercalation activity.

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

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

Synthesis and pharmacological evaluation of novel 9- and 10-substituted cytisine derivatives. Nicotinic ligands of enhanced subtype selectivity.

We report the synthesis and pharmacological properties of several cytisine derivatives. Among them, two 10-substituted derivatives showed much higher selectivities for the alpha4beta2 nAChR subtype in binding assays than cytisine. The 9-vinyl derivative was found to have a very similar agonist activity profile to that of cytisine.

Synthesis and structure-activity relationships of 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine analogues as potent, noncompetitive metabotropic glutamate receptor subtype 5 antagonists; search for cocaine medications.

Recent genetic and pharmacological studies have suggested that the metabotropic glutamate receptor subtype 5 (mGluR5) may represent a druggable target in identifying new therapeutics for the treatment of various central nervous system disorders including drug abuse. In particular, considerable attention in the mGluR5 field has been devoted to identifying ligands that bind to the allosteric modulatory site, distinct from the site for the primary agonist glutamate. Both 2-methyl-6-(phenylethynyl)pyridine (MPEP) and its analogue 3-[(2-methyl-4-thiazolyl)ethynyl]pyridine (MTEP) have been shown to be selective and potent noncompetitive antagonists of mGluR5. Because of results presented in this study showing that MTEP prevents the reinstatement of cocaine self-administration caused by the presentation of environmental cues previously associated with cocaine availability, we have prepared a series of analogues of MTEP with the aim of gaining a better understanding of the structural features relevant to its antagonist potency and with the ultimate aim of investigating the effects of such compounds in blunting the self-administration of cocaine. These efforts have led to the identification of compounds showing higher potency as mGluR5 antagonists than either MPEP or MTEP. Two compounds 19 and 59 exhibited functional activity as mGluR5 antagonists that are 490 and 230 times, respectively, better than that of MTEP.

Identification of HDAC6-Selective Inhibitors of Low Cancer Cell Cytotoxicity.

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform-selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first-generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell-cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole-based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.

Methoxyphenylethynyl, methoxypyridylethynyl and phenylethynyl derivatives of pyridine: synthesis, radiolabeling and evaluation of new PET ligands for metabotropic glutamate subtype 5 receptors.

We have synthesized three different PET ligands to investigate the physiological function of metabotropic glutamate subtype 5 receptors (mGluR5) in vivo: 2-[(11)C]methyl-6-(2-phenylethynyl)pyridine ([(11)C]MPEP), 2-(2-(3-[(11)C]methoxyphenyl)ethynyl)pyridine ([(11)C]M-MPEP) and 2-(2-(5-[(11)C]methoxypyridin-3-yl)ethynyl)pyridine ([(11)C]M-PEPy). [(11)C]Methyl iodide was used to label the compounds under basic conditions, and a Pd(0) catalyst was applied to label [(11)C]MPEP in a Stille coupling reaction. In vivo microPET imaging studies of the functional accumulation of radiolabeled ligands were conducted in 35 rats (Sprague-Dawley, 8 weeks old male, weight of 300 g). Specific binding was tested using pre-administration of unlabeled mGluR5 antagonist 2-methyl-6-(2-phenylethynyl)pyridine (MPEP) (10 mg/kg iv 5 min before radioactivity injection). In the radiolabeling of [(11)C]MPEP, [(11)C]M-MPEP and [(11)C]M-PEPy, a specific radioactivity of 700-1200 mCi/micromol and over 97% radiochemical purity were obtained. The microPET studies showed these three radiolabeled mGluR5 antagonists having the highest binding in the olfactory bulb followed by striatum, hippocampus and cortex. Pre-administration of the mGluR5 antagonist MPEP induced a 45.1% decrease in [(11)C]MPEP binding, a 59.7% decrease in [(11)C]M-MPEP binding and an 84.6% decrease in [(11)C]M-PEPy binding in the olfactory bulb at 5 min. The feasibility of synthesizing high-affinity and high-selectivity ligands for mGluR5 receptors and their suitability as PET imaging ligands for mGluR5 receptors in vivo are demonstrated.

Synthesis and preliminary biological evaluation of 3-[(18)F]fluoro-5-(2-pyridinylethynyl)benzonitrile as a PET radiotracer for imaging metabotropic glutamate receptor subtype 5.

The metabotropic glutamate receptor subtype 5 (mGluR5) has been reported to be implicated in various neurological disorders in the central nervous system. To investigate physiological and pathological functions of mGluR5, noninvasive imaging in a living body with PET technology and an mGluR5-specific radiotracer is urgently needed. Here, we report the synthesis of 3-[(18)F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([(18)F]FPEB) through a convenient thermal reaction as a highly specific PET radiotracer for mGluR5. The precursor and standard compounds were prepared by a coupling reaction catalyzed by palladium. Radiosynthesis of [(18)F]FPEB was performed using nitro as a leaving group replaced by [(18)F]fluoride under conventional heating condition. Biodistribution, metabolite, and microPET studies were performed using Sprague-Dawley rats. Upto 30 mCi of [(18)F]FPEB was obtained with a radiochemical yield of 5% and a specific activity of 1900 +/- 200 mCi/mumol at the end of syntheses. Biodistribution showed rapid clearance from the blood pool and fast and steady accumulation of radioactivity into the brain. Metabolite studies indicated that only 22% of [(18)F]FPEB remained in the blood system 10 min after administration, and that a metabolite existed which was much more polar than the parent tracer. MicroPET studies demonstrated that [(18)F]FPEB accumulated specifically in mGluR5-rich regions of the brain such as striatum and hippocampus, and that blockade with 2-methyl-6-(2-phenylethynyl)pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) substantially reduced the activity uptake in these regions. Selectivity was investigated by blockage with 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-caroxamide (YM-298198), a specific antagonist for mGluR1. [(18)F]FPEB was prepared conveniently and showed high specificity and selectivity toward mGluR5. It possesses the potential to be used in human studies to evaluate mGluR5 functions in various neurological disorders.