Biological tests

As already said in Paragraph 4.1.2, when TTR is subjected to mutation, the tetrameric quaternary structure is destabilized and so the unfolding and the amyloid deposit formation is triggered. Since classical inhibitors usually stabilize the mutant TTR quaternary structure, the degree of oligomerization is a reliable index of stabilization efficiency and so, for this reason, separation techniques on molecular weight must be employed. One of the most famous is SDS-PAGE/Western-Blotting (or immunoblotting)⁸⁸, which consists in the immobilization on membrane and identification, with a specific antibody, of a specific antigene (or protein), present in a complex mixture of antigens (or proteins) upon a molecular weight-based

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separation in polyacrylamide gel (alternatively dot blot or slot blot, not based on molecular weight separation, but just on antibody/antigene selectivity).

The method was created by George Stark at Standford University. The name comes ironically from “Southern blot”, technique for DNA detection previously invented by Edwin Southern.

These techniques, along with Northern blot (for RNA detection), are very important and basilar techniques commonly used in biochemistry, immunogenetics and molecular biology.

The SDS-PAGE/Western-Blotting is made of four steps:



 SDS-PAGE: protein separation by electrophoresis on polyacrylamide gel with sodium dodecylsulphate

 Western Blotting: transfer of proteins separated on gel onto a membrane, exposition of the membrane with antibody selective for the protein of interest (primary antibody), exposition of the membrane with labelled-antibody selective for primary antibody (secondary antibody), detection

 Densitometric analysis of pictures

The extraction step is composed of two phases: cellular lysis and protein isolation. The cellular lysis phase is essential to break biological membrane release macromolecules in buffer.

Generally, upon appropriate preparation of tissues or cell culture by mechanical breaking with shaker (large volume), homogeneizator (small volume) or sonication, cytosolic proteins are collected as supernatant by centrifugation. To increase lysis and preserve the integrity of proteins by modification, degradation and unfolding, a buffer of salts, detergents and phosphatase and protease inhibitors is employed along with a working temperature of 4 °C, which limits side-reactions. If the target is a particular organelle protein, the supernatant is disposed and the pellet is treated with different biochemical and mechanical strategies using suitable cut-off filters. The protein supernatant is then quantified by spectrophotometric methods in order to determine the volume of protein solution to be used in the next analysis.

Figure 4.3.5.1: Schematic representation of an SDS-PAGE development

SDS-PAGE, because of its denaturing conditions, is the most favourite technique for protein purification just according to their molecular weight. In fact, here it is used sodium dodecyl sulphate, which coats proteins linearizing them. Since these aggregates are negatively-charged for sulphate groups, the actual protein charge is essentially shielded and so the separation can

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be done just on molecular weight, where small proteins will run faster than the bigger ones. The electrophoretic run is done down to a polyacrylamide gel towards the positive anod inside a cassette prefilled with electrolytic buffer (generally TrisGly-pH 8.3). The gel is made in situ by polymerization of acrylamide and bis-acrylamide in Tris HCl buffer using APS (ammonium persulphate) as a cross-linking agent and TEMED (tetramethylenenediamine) as catalyst.

Actually the generation of gel is made in a two-step process. In fact, first of all, a running gel is made taking the polymerization buffer at pH 8.8, which guarantees a type of gel with small pores appropriate for protein separation (the acrylamide percentage varies from 7.5% to 15%

according to protein dimensions), meanwhile on the top of it is gelified a stacking gel with 4%

acrylamide at pH 6.8, characterized by a very small cross-linking degree, appropriate for sample deposition. To facilitate the deposition, in sample buffer is added glycerol, which, being denser than buffer, drag the protein down to wells and prevents the mixing with the surrounding buffer. Beyond SDS, DTT and β-mercaptoethanol as denaturating agents, in sample buffer is present bromophenol blue, a negatively-charged dye, which, essentially behaving like the smallest protein in the sample, is used to track and monitor the electrophoretic run. Before loading into wells, lysate is heated up at 100 °C for 5 minutes into sample buffer to denaturate effectively the proteins to get them ready for electrophoresis. Once loaded the samples, proteins are firstly absorbed in stacking gel up to the interphase with running gel using low voltage (typically 60 V) and then it is raised up to 100V-200V to start the separation. The mass of protein bands can be easily compared with those coloured of first well (“protein ladder”) which belongs generally to a standard of pre-stained proteins of known molecular weight. To enhance protein bands the gel is generally stained with different agents such as Coomassie blue or Zinc silver. This classical staining is employed generally when there is no problem of sensitivity and when the sample is not overcrowded, otherwise Western Blotting technique is required for satisfying results.

Figure 4.3.5.2: Schematic representation of Wester Blotting Transfer

Here, the gel is put in contact with an adsorbent membrane (Nitrocellulosa or Polivinilidenfluoruro), both of them enclosed between pairs of filter paper and sponges. The voltage polarity is negative to the gel and positive to the membrane. The power is switched on, the protein bands start to migrate to anod till they are captured by the membrane. This preliminary transfer is necessary to get proteins easily-accessible to next immunoassay. At the end of the blotting, since this membrane is sensible to all type of proteins, a blocking step with fat dry milk or purified BSA in Tris buffer is required to remove non-specific antibody background binding (0.5 - 5 ug/ml). Then the adsorbed proteins are rocked in a solution of antibody (primary antibody) specific to epitope present into protein of interest.

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Upon repetitive washing with a mild detergent (TBST/PBST, tween 20 or triton x-100) in blocking solution, a further membrane wash is made with an antibody (secondary antibody) specific for the primary antibody used in the assay.

Figure 4.3.5.3: Examples of the most common types of immunorevelation.

This last antibody is generally labelled with different tags such as fluorophores, radioactive iodine atoms or reporter enzymes to get bands detected with UV/Vis light emission, X-ray emission or chemiluminescence (e.g horseradish peroxidases). In the case of UV-vis light, the detection is realized with CCD cameras, which capture the image of Western Blot, which in turn can be subjected to a densitometric analysis to assess the protein concentration for each band.

In the common lab routine UVvis methods are preferred to X-ray methods since much safer.

In Berni’s laboratory in the perspective of using 3-deoxytolcapone (65) as TTR stabilizer for ATTR pharmacological therapy, a Western Blotting method was optimized to qualitatively determine its affinity for TTR in plasma samples and at the same time give a proof of its stabilizing properties on the tetrameric TTR under partially denaturing conditions. In fact in order to replicate in plasma sample the high concentrations of monomer typical of FAP, plasmatic TTR was incubated with compound 65 in presence of 4M urea. These conditions provide the dissociation of the tetramer into monomers without affecting their folding, so the resulting monomer concentration was assumed representative of the fraction of denatured protein present in the sample. Operatively compound 65, dissolved in DMSO, was incubated for 2h at 20°C in diluted plasma and then a volume of 8M urea was added in to get drug final concentrations of 15M, 7.5M, 4M and 2M. The mixture was finally left incubating for further 18 h at 20 °C. Upon SDS-PAGE and blotting, realized as described before, the immunodetection was realized with rabbit anti-human TTR polyclonal Ab (Dako) as primary Ab, and anti-rabbit Ab labelled with Dylight 680 (SERACARE) as secondary Ab, where the images were recorded using an Odyssey Image System (LI-COR).

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Figure 4.3.5.4. SDS-PAGE/Western Blotting of several drugs and potential inhibitors towards WT-TTR in plasma samples in denaturing conditions. In all SDS-PAGEs DMSO or PLASMA+UREA lanes were taken as negative controls, meanwhile Tafamidis and Tolcapone were taken as positive controls. DS108 corresponds to 3-deoxytolcapone 65. Different concentrations of drugs were tested in the assay: 15M (A), 7.5M (B) and 2-4M (C). In panel D molecular structures are illustrated of Flurbiprofene, CHF5074 and DS113, an intermediate for sulfation of 3-deoxytolcapone.

As it can be seen in Figure 4.3.5.4, all SDS-PAGE lanes are made of (from the bottom to the top):

i) a first low band corresponding to the monomer, ii) an intermediate band, particularly evident upon treatment of TTR with DMSO and corresponding to the dimer and iii) a group of intense bands due to trimer, tetramer and other aggregates at higher MW of TTR. It is important to compare the monomer band in the different experiments, since its intensity represents a measurement of how much stabilizing is the molecule for the tetramer: the less intense, the better. As it can be seen by Figure 4.3.5.4A, compound DS113 (precursor of sulphated 3-deoxytolcapone), diflunisal, flurbiprofene and CHF5074 are increasingly worse stabilizers (bands progressively more intense), while tafamidis, tolcapone, 3-O-methyl-tolcapone and compound 65 (DS108) showed to be very interesting stabilizers (bands faded), but at 15 M concentration no difference among them is observed. Since neither at concentration of 7.5 M our compound showed difference respect to tolcapone in stabilizing TTR tetramer (Figure 4.3.5.4B), we moved

A B

C D

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to concentrations of 4 and 2 M. Here (Figure 4.3.5.4C) discrimination is clearly seen in different band intensities. In fact, in plasma compound 65 (DS108) is clearly more selective to plasmatic WT-TTR than Tafamidis, which is the preferred drug used for the treatment of FAP nowadays. For this reason along with the fact that compound 65 includes deletion of the glucuronidation-affected OH, it represents a good alternative to Tafamidis. Unfortunately, compound 65 is less selective to WT-TTR than parent drugs tolcapone and methyltolcapone.

Considering though that methyltolcapone is subjected to demethylation in hepatocytes being transformed in tolcapone (with all related problems inherent to hepatic overwork), our 3-deoxytolcapone is a promising tool for the treatment of FAP. In future this molecule will be studied in pharmacokinetic experiments in the hope to increase the half-life of the active substance circulating in blood.

In an analogous way to 3-deoxytolcapone, these BiochaninA derivatives were tested too in SDS-PAGE/Western Blotting. As it can be seen in Figure 4.3.5.5, compound 67 (DS114), 68 (DS120), 69 (DS121) and 70 (DS124) seem to be not selective to WT-TTR in serum samples.

Figure 4.3.5.5: Western Blots of compound 67 (DS114), 68 (DS120), 69 (DS121) and 70 (DS124) with hTTR in plasma samples in denaturing conditions. Different drug concentrations were used in the assay: 7.5 M (A) and 15 M (B).

They, in fact, are characterized by a band corresponding to the monomer intense as much as for DMSO treatment, which is representative of actual monomeric TTR concentration observable in the disease. With this data, we cannot say anything about their affinities towards single TTR, but even if they were very efficient, they are totally useless for an actual therapy against amyloidogenic disease. Probably, the sulfate modification makes the molecule too bulky and non-electronically complementary to the active site.

In the whole discussion, we talked about selectivity and not affinity because the matrix is a complex mixture of several proteins. This means that these molecules in plasma can be either sequestrated or not by other targets so the bioavailability can be changed. In other words, Western Blot in plasma samples is representative of the effects of the molecules on the whole matrix without taking into account the single interaction.

A B

A B

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