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Design, Synthesis, and Evaluation of Hydroxamic Acid-Based Molecular Probes for In Vivo Imaging of Histone Deacetylase (HDAC) in Brain
Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging.Chemistry and Chemical Biolog
Original Article Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain
Abstract: Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging
Activation of Progesterone Receptor by ATP
Progesterone-receptor complex from freshly prepared hen oviduct cytosol acquired the ability to bind to
isolated nuclei, DNA-cellulose and ATP-Sepharose when incubated with 5-10 mM ATP at 4°C. The extent of
this ATP-dependent activation was higher when compared with heat-activation achieved by warming the progesterone-
receptor complex at 23 °C. The transformation of progesterone-receptor complex which occurred in a
time-dependent manner was only partially dependent on hormone presence. The ATP effect was selective in
causing this transformation whereas ADP, AMP and cAMP failed to show any such effect. The non-hydrolizable
analogs of ATP, adenosine 5'-[a,/3-methylene]triphosphate and adenosine 5-[/l,y-imido]triphosphate were also
found to be ineffective. Presence of 10 mM sodium molybdate blocked both the ATP and the heat-activation of
progesterone-receptor complex. Mn" or Mg` had no detectable effect on the receptor activation but the presence
of Ca" increased the extent of ATP-activation slightly. EDTA presence (> 5 mM) decreased the extent of
receptor activation by about 40 % and was, therefore, not included in the buffers used for activation studies.
Divalent cations were also ineffective when tested in the presence of 1- 5 mM EDTA. The
properties of progesterone-receptor complex remained intact under the above conditions when analyzed for
steroid-binding specificity and Scatchard analysis. However, the ATP-activated progesterone-receptor complex
lost the ability to aggregate when tested on low-salt sucrose gradients. ATP was equally effective in activating
the rat-uterine estradiol-receptor complex at 4 "C and influenced the transformation of 4-S receptor form into
a 5-S form when analyzed on sucrose gradients containing 0.3 M KCI. The presence of ATP also increased the
rate of activation of progesterone-receptor complex at 23 °C. These findings suggest a role for ATP in receptor
function and offer a convenient method of studying the process of receptor activation at low temperature and
mild assay conditions
In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography
The Discovery, Preclinical, and Early Clinical Development of Potent and Selective GPR40 Agonists for the Treatment of Type 2 Diabetes Mellitus (LY2881835, LY2922083, and LY2922470)
The
G protein-coupled receptor 40 (GPR40) also known as free fatty
acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet β-cells
and responds to endogenous fatty acids, resulting in amplification
of insulin secretion only in the presence of elevated glucose levels.
Hypothesis driven structural modifications to endogenous FFAs, focused
on breaking planarity and reducing lipophilicity, led to the identification
of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40
agonists with unique pharmacology, selectivity, and pharmacokinetic
properties. Compounds <b>1</b> (LY2881835), <b>2</b> (LY2922083),
and <b>3</b> (LY2922470) demonstrated potent, efficacious, and
durable dose-dependent reductions in glucose levels along with significant
increases in insulin and GLP-1 secretion during preclinical testing.
A clinical study with <b>3</b> administered to subjects with
T2DM provided proof of concept of <b>3</b> as a potential glucose-lowering
therapy. This manuscript summarizes the scientific rationale, medicinal
chemistry, preclinical, and early development data of this new class
of GPR40 agonists