Posts Tagged ‘COX 1 & 2 inhibitors’

Reporter and curator: Larry H. Bernstein, MD, FCAP that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity

Transthyretin is a small protein with a half-life of < 48 hours, synthesized by the liver, and a major transport protein for thyroxin.  There are 80 variants known, and some variants that occur in the Portuguese, a small section of Japan, Sweden, and Brazil, are associated will primary amyloidosis, the only cure for which is liver transplantation.  It causes fibrillary inclusions in the heart, but also affects the autonomic nervous system.  Some of the major work on this has been done for many years in the laboratory of   Jeffery W. Kelly, at the Skaggs Institue for Chemical Biology, the Scripps Research Institute.  A recent publication is of considerable interest.

Potent Kinetic Stabilizers that Prevent Transthyretin-mediated Cardiomyocyte Proteotoxicity

 Mamoun M. Alhamadsheh1,6,7, Stephen Connelly2,7, Ahryon Cho1, Natàlia Reixach3, Evan T. Powers3,4,5, Dorothy W. Pan1, Ian A. Wilson2,5, Jeffery W. Kelly3,4,5, and Isabella A. Graef1,*
Sci Transl Med. Author manuscript; available in PMC 2012 August 24.
1Department of Pathology, Stanford University Medical School, Stanford, California, USA
2Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
4Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA
5The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
6Department of Pharmaceutics & Medicinal Chemistry, University of the Pacific, Stockton, California, USA


The V122I mutation that alters the stability of transthyretin (TTR) affects 3–4% of African Americans and leads to amyloidogenesis and development of cardiomyopathy. In addition, 10–15% of individuals over the age of 65 develop senile systemic amyloidosis (SSA) and cardiac
TTR deposits due to wild-type TTR amyloidogenesis. As no approved therapies for TTR amyloid cardiomyopathy are available, the development of drugs that prevent amyloid-mediated cardiotoxicity is desired. To this aim, we developed a fluorescence polarization-based HTS screen,
which identified several new chemical scaffolds targeting TTR. These novel compounds were potent kinetic stabilizers of TTR and
  • prevented tetramer dissociation,
  • unfolding and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR.
High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. Our study also showed that these compounds effectively inhibited the proteotoxicity of V122I-TTR towards human cardiomyocytes.
Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is one of the best known inhibitors of TTR aggregation, and may be promising leads for the treatment and/or prevention of TTR-mediated cardiomyopathy.

Author Contributions:

M.M.A. designed and performed most experiments, S.C. performed crystallographic structure determination, A.C peformed the serum TTR stabilization. N.R. performed the cell-based assays.   E.T.P. analyzed the ITC data. D.W.P. helped with probe synthesis. I.A.W. supervised the crystallographic work. J.W.K. supervised the work, S.C., N.R., I.A.W. and J.W.K. edited the paper. I.A.G supervised the work, M.M.A. and I.A.G prepared the manuscript.


The misassembly of soluble proteins into toxic amyloid aggregates underlies a large number of human degenerative diseases (1–3). TTR is one of more than 30 human amyloidogenic proteins whose misassembly can cause
  • a variety of degenerative gain-of-toxic-function diseases.
TTR is a tetrameric protein (54 kDa), secreted from the liver into the blood where, using orthogonal sites,
  • it transports thyroxine (T4) and
  • holo-retinol binding protein (4).
However, 99% of the TTR T4 binding sites remain unoccupied in humans
  • owing to the presence of two other T4 transport proteins in blood (3).
Familial TTR amyloid diseases, which are associated with one of more than 80 mutations in the TTR gene, include
  • the systemic neuropathies (familial amyloid polyneuropathy [FAP]),
  • cardiomyopathies (familial amyloid cardiomyopathy [FAC]), and
  • central nervous system amyloidoses (CNSA) (5–8).
Cardiac amyloidosis is most commonly caused by
  • deposition of immunoglobulin light chains or
  • TTR in the cardiac interstitium and conducting system.
It is a chronic and progressive condition, which can lead to arrhythmias, biventricular heart failure, and death (8–10). Two types of TTR-associated amyloid cardiomyopathies are clinically important.
  1. Wild-type (WT) TTR aggregation underlies the development of senile systemic amyloidosis (SSA). Cardiac TTR deposits can be found in 10 to 15% of the population over the age of 65 at autopsy (10,11). Many of these patients are asymptomatic, but there is little doubt that SSA is an underdiagnosed disease.
  2. In addition, a number of TTR mutations, including V122I, lead to amyloidogenesis and familial amyloid cardiomyopathy (FAC) (12–15). Population studies show that the V122I mutation is found in 3–4% of African Americans (~1.3 million people) and contributes to the increased prevalence of heart failure among this population segment (14,15).

The mutant TTR allele behaves as an autosomal dominant allele with age-dependent penetrance and

  • the frequency of cardiac amyloidosis from TTR in African-American individuals above age 60 is four times that seen in Caucasian-Americans of comparable age.
All of the TTR mutations associated with familial amyloidosis decrease tetramer stability, and
  • some decrease the kinetic barrier for tetramer dissociation (3, 16).
  • The latter is important because tetramer dissociation is the rate-limiting step in the TTR amyloidogenesis cascade (3).

Kinetic stabilization of the native, tetrameric structure of TTR by

  • interallelic trans suppression (incorporation of mutant subunits that raise the dissociative transition state energy) prevents
    1. post-secretory dissociation and aggregation, as well as the related disease 
    2. familial amyloid polyneuropathy (FAP), by slowing TTR tetramer dissociation (17).
Occupancy of the TTR T4 binding sites with rationally designed small molecules is known to stabilize the native tetrameric state of TTR over the dissociative transition state,
  • raising the kinetic barrier,
  • imposing kinetic stabilization on the tetramer and
  • preventing amyloidogenesis (3, 16, 18).
Previous studies have focused on rational ligand design and as a result
  • most of the TTR stabilizers reported to date are halogenated biaryl analogues of T4,
  • many resembling non-steroidal anti-inflammatory drugs (NSAIDs).
Some of these compounds, such as the NSAID diflunisal, which is currently tested in clinical trials in FAP patients for its efficacy to ameliorate
  • peripheral neuropathy resulting from TTR deposition, (19) have anti-inflammatory activity (20, 21).
The pharmacological effects of NSAIDs are due to inhibition of cyclo-oxygenase (COX) enzymes (22). Inhibition of COX-1 can produce side effects such as
  • gastrointestinal irritation, leading to ulcers and bleeding (23).
Inhibition of COX-2 has been associated with an
  • increased risk of severe cardiovascular events, including heart failure,
  • particularly in patients with preexisting cardiorenal dysfunction (20, 21, 24, 25).
Therefore, heart and kidney impairment are exclusion criteria for participation of patients in the diflunisal clinical trials to treat TTR-mediated FAP (19). Genomic variations can
  • increase the sensitivity of individuals to adverse side effects of NSAIDs.
Serum concentrations of NSAIDs depend on CYP2C9 and/or CYP2C8 activity. CYP2C9 polymorphism might play a significant role in the profile of adverse side effects of NSAID and alleles that affect the activity of CYP2C9 are found at different frequency in subjects of Caucasian, African or Asian descent (26, 27). Hence, the long-term therapy with drugs that have inhibitory effect on COX activity to prevent TTR aggregation is especially problematic in patients who suffer from TTR-mediated cardiomyopathy. The design and development of drugs to treat/prevent FAC or SSA thus presents the challenge
  1. not only to find compounds with a greater variety of chemical scaffolds that accomplish stabilization, but
  2. do so without the adverse side effects due to inhibition of COX activity.
 For these reasons, the development of a rapid and robust screen for compounds that bind to and stabilize TTR could be useful. To date, no high-throughput screening (HTS) methodology is available for the discovery of TTR ligands (28,29). Therefore, we developed a versatile
  • fluorescence polarization (FP) based HTS assay that can detect
  • binding of small molecules to the T4 binding pocket of TTR under physiological conditions.


Design and synthesis of the TTR FP probe

FP is used to study molecular interactions by monitoring changes in the apparent size of a fluorescently labeled molecule. Binding is measured by an increase in the FP signal, which is proportional to the decrease in the rate of tumbling of a fluorescent ligand upon association with macromolecules such as proteins (Fig. 1A). To synthesize a fluorescent TTR ligand 1, we initially started with the NSAID diflunisal analogue 2 (Fig. 1B) (30). The product of attaching a linker to 2, compound 3, had very low binding affinity to TTR (Kd1 >3290 nM, fig. S1A and fig. S1B).
The crystal structure of the diclofenac analog 4 showed that
  • the phenolic hydroxyl flanked by the two chlorine atoms is oriented out of the binding pocket into the solvent (31).
  • We reasoned that attaching a PEG amine linker to the phenol group of 4 would generate compound 5 which would bind to TTR (Fig. 1B and fig. S1C)

5 was coupled to fluorescein isothiocyanate (FITC) to produce the FITC-coupled TTR FP probe (1, Fig. 1B). The binding characteristics of the probe (Kd1 = 13 nM and Kd2 = 100 nM) were assessed with ITC (Fig. 2A).

Evaluation of the FP assay

The binding of 1 to TTR was evaluated to test its suitability for the FP assay with a standard saturation binding experiment. A fixed concentration of probe 1 (0.1 μM) was incubated with increasing concentrations of TTR (0.005 μM to 10 μM) and the formation of 1•TTR complex was quantified by the increase in FP signal (excitation λ 485 nm, emission λ 525 nm) relative to the concentration of TTR (Fig. 2B). The fluorescence polarization increased with the concentration of TTR until saturation was reached. A large dynamic range (70 – 330 mP) was measured for the assay. To validate the FP assay, we tested known TTR binders in a displacement assay (for detailed information see Supplemental Material). Compound 2 (Kapp = 231 nM, R2 = 0.997), Thyroxine (T4) (Kapp = 186 nM, R2 = 0.998) and diclofenac (Kapp = 4660 nM, R2 = 0.999) decreased the FP signal in a dose- dependent  manner  (Fig. 2C,  fig. S2B and S2C). The FP assay is a competitive displacement assay and therefore it provides apparent binding constants (Kapp). However, these apparent binding constants correlate well with the data obtained by ITC which measures direct interactions in solution and gives an actual (Kd) value.

  Adaptation of the FP assay for HTS

Next, we optimized the FP assay for HTS and screened a ~130,000 small molecule library for compounds that displaced probe 1 from the T4 binding sites of TTR. The FP assay was performed in 384-well plates with low concentrations of probe 1 (1.5 nM) and TTR (50 nM) in a 10  μL assay volume.  Detergent (0.01% Triton X-100) was added to the assay buffer to avoid false positive hits from aggregation of the small molecules. The assay demonstrated robust performance, with a, large dynamic range (~70–230 mP) and a Z′ factor (32, 33) in the range of 0.57–0.78 (fig. S3A and S3B).

Hits were defined as compounds, which resulted in at least 50% decrease in FP and demonstrated relative fluorescence between 70 and 130%. Many fluorescence quenchers and enhancers, which have less than 70% and greater than 130% total fluorescence relative to a control (compound without TTR), were excluded from the hit list. The excluded compounds have native fluorescence that is similar to fluorescein, which would interfere with the FP measurements and result in false positive hits. Two hundred compounds were designated as positive hits (0.167% hit rate). The top 33 compounds (compounds with lowest FP IC50) were assayed in a 10-point duplicate dose-response FP assay and displayed an IC50 (concentration that resulted in 50% decrease in the FP signal) between 0.277 and 10.957 μM (table S2).

Validation of the HTS hits

The top 33 compounds were retested with the FP assay (table S2) and with surface plasmon resonance (SPR) as another independent biophysical method. Solutions of the 33 hits were passed over immobilized, biotinylated TTR on a streptavidin coated chip. The binding of a small molecule to TTR on the sensor chip produces a SPR response signal (RU). The RU signal after addition of the top 33 compounds was measured and compared to a negative, solvent only, control. All compounds identified by the screen as hits were confirmed as TTR binders using SPR (fig. S4). We also found known TTR binders, such as NSAIDs (diclofenac, meclofenamic acid, and niflumic acid) and isoflavones (apigenin) in our screen (3, 34) (table S2). Among the best ligands (Fig. 2D) were the NSAID, niflumic acid, two catechol-O-methyl-tranferase (COMT) inhibitors, 3,5-dintrocatechol and Ro 41-0960 (35) and a number  of compounds   not previously known to bind to TTR. The chemical structures of these ligands were confirmed by 1H NMR and high-resolution mass spectrometry(HRMS) and the chemical purity was determined to be >95% (fig. S5).

Inhibition of TTR amyloidogenesis by the HTS hits

To test whether the new TTR ligands (7.2 μM) could function as kinetic stabilizers, we measured their ability to inhibit TTR (3.6 μM) amyloidogenesis at 72 hrs at pH 4.4 (fig. S6) (29). All 33 compounds inhibited TTR aggregation (<50% fibril formation, table S2). Of these, 23 were very good (<20% fibril formation) and 11 were excellent (<2% fibril formation) TTR kinetic stabilizers (Fig. 3A). All of the potent TTR stabilizers, except niflumic acid, and the two COMT inhibitors 3,5-dintrocatechol and Ro 41-0960, were chemical entities with no previously reported biological activity. Since occupancy of only
one T4 binding site within TTR is sufficient for kinetic stabilization of the tetramer (3), we tested the most potent ligands at substoichiometric concentrations (2.4 fold molar excess of TTR relative to ligand) in a kinetic aggregation assay monitored over 5 days (Fig. 3B). Under these conditions ligands 7, 14, 15 and Ro 41-0960 dramatically slowed fibril formation and outperformed the known TTR stabilizer, diclofenac, which blocked only ~55% of TTR aggregation.

Evaluating the TTR ligands for COX-1 enzymatic inhibition and binding to thyroid hormone receptor

A successful clinical candidate against TTR amyloid cardiomyopathy should have minimal off-target toxicity due to the potential need for life-long use of these drugs. Specifically, the TTR ligands should exhibit minimal binding to COX and the nuclear thyroid hormone receptor (THR). Inhibition of COX is contraindicated for treating FAC patients, since COX inhibition can not only lead to renal dysfunction and blood pressure elevation, but may precipitate heart failure in vulnerable individuals (20, 21, 24, 25). Therefore, the most potent TTR ligands were evaluated for their ability to inhibit COX-1 activity, as well as, for binding to THR, in comparison with the NSAID niflumic acid. Although niflumic acid exhibited substantial (94%) COX-1 inhibition, three of the 12 new compounds evaluated (7, 6 and 10) displayed less than 1% inhibition of COX-1. Only one ligand (compound 8) showed significant (58%) and two compounds (6 and 10) minor (5%) binding to THR (Fig. 3C).

Characterization of the binding energetics to TTR

Many reported TTR ligands, including T4, bind TTR with negative cooperativity, which appears to arise from subtle conformational changes in TTR upon ligand binding to the first T4 site (3, 16, 36). We used ITC to determine the binding constants and to evaluate cooperativity between the two TTR T4 sites (Fig. 2A, Fig. 4A, Fig. 4B and fig. S1 and fig. S7). The ITC data for compounds 1, 7, 14, and Ro 41-0906 binding to TTR were fit to a two-site binding model and show that these potent ligands bind TTR with low nanomolar affinity. The dissociation constants for these ligands indicated that they bound TTR with negative cooperativity (table S3). Analysis of the free energies associated with ligand binding to TTR indicates that binding was driven both by burial of the hydrophobic ligand in the TTR binding site (which leads to the favorable binding entropies) and specific ligand-TTR interactions (which leads to the favorable binding enthalpies) (Fig. 2A, Fig. 4A, Fig.4B, and fig. S7B) (37). The binding of compounds 7 (Kd1 = 58 nM and Kd2 = 500 nM) and 14 (Kd1 = 26 nM and Kd2 = 1800 nM) to TTR did not cause major conformational changes to the TTR tetramer structure (Fig. 5).
Remainder of document is found at publication site, including Figures.

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