ITMI20072323A1 - PEPTIDES INHIBITORS OF TUBULINE POLYMERIZATION - Google Patents

Description

"PEPTIDES INHIBITORS OF TUBULIN POLYMERIZATION"

The invention provides tubulin polymerization inhibitors.

Background of the invention

Tubulin is composed of a heterodimer formed by two proteins (alpha and beta tubulin) which polymerize giving rise to αβαβαβ sequences ... These sequences form filaments that assemble in such a way as to form microtubules.

In the polymerization process, an αβ unit joins with the subsequent αβ unit through a series of complex non-covalent interactions on an interaction surface equal to about 3000 A.

Tubulin is currently one of the most interesting targets of antimitotic drugs capable of blocking the growth of cancer cells. Several molecules capable of modifying its polymerization dynamics, mainly of natural origin, are currently in use. In recent years, a broad line of research has been developed aimed at identifying small molecules capable of effectively binding to protein regions that act as interfaces in protein-protein interactions to prevent protein aggregation or polymerization. This objective is of primary interest to modulate all those processes where the interactions between proteins play a fundamental role.

Polymerization of tubulin leads to the formation of microtubules which are involved in multiple cell functions, including cell division processes. The growth and rupture of microtubules is a process characterized by a very complex kinetics and the interaction with this process can lead to a stabilization or destabilization of the microtubules with heavy repercussions on the cell cycle.

In fact, several anticancer drugs use this process to block the growth of cancer cells.

Several compounds mostly of natural origin, such as Vinca alkaloids, colchicinoids and taxane derivatives, interact with tubulin in different sites of its structure.

These compounds have severe side effects, due both to the intrinsic characteristics of the drugs and to the additives that must be used for their administration. Furthermore, being substances of natural origin, their supply or synthesis is a laborious process.

The combined use of different drugs can allow their use in lower doses, thus reducing side effects. Also from this point of view, therefore, the knowledge of new tubulin polymerization inhibitors is therefore particularly important and desirable.

Due to the complexity of each tubulin heterodimer and to the fact that the interaction region between two successive units covers an area of about 3000 A 'therefore characterized by a large number of complex interactions, the identification of suitable amino acid sequences capable of carrying out an inhibitory action presents considerable problems and difficulties, as the number of theoretically selectable peptides is enormous. It should also be noted that in this case the target of the inhibitor is a surface between two proteins and there is a vast literature on the difficulties of developing small molecules that "mimicking" a protein can actually block the interaction between protein surfaces. This is mainly due to two reasons: a) the interproteic surface did not evolve to interact with small molecules; b) although similarities between surfaces of different proteins have begun to be highlighted, there is still not a sufficient amount of information to establish binding analogies and therefore each interproteic surface represents a case in itself.

Summary of the invention

The present invention relates to peptides with sequences derived from tubulin which inhibit the polymerization process thereof.

The peptides of the invention have the following sequences:

Ser-Pro-Lys-Val-Ser-Asp-Thr-Val-Val (Peptide 1)

Ala-Trp-Xaa-Pro-Thr-Gly-Phe-Lys-Val-Gly (Peptide 2; Xaa is Cys or Ala);

Phe-Arg-Arg-Lys-Ala-Phe-Leu-His-Trp-Tyr-Thr-Gly (Peptide 3).

Said sequences are subsequences of the β (peptide 1 and 3) and a (peptide 2) units of tubulin, with the sole exception of the Alai residue of peptide 2 (which replaced the Asp residue present in the original sequence). The three peptides are found at the interface between the two units a and β as shown by the crystallographic structure deposited in the Protein Data Bank (structure lTUB.pdb). The invention also includes the derivatives of the said peptides obtained by replacing the natural amino acids with the corresponding amino acids of the D series and / or the derivatization of the hydroxide, thiol functional groups or the basic functional groups of serine, threonine, tyrosine, cysteine, arginine, lysine and / or functionalization of terminal N and C groups, for example with acetate to amide groups. The invention also includes derivatives obtained by retro-inversion of one or more peptide bonds, according to known techniques that allow the peptides to be stabilized against hydrolytic enzymes, thereby improving their pharmacokinetic characteristics. The present invention also includes derivatives or analogs of said peptides obtained with conservative mutation of amino acids, typically from one to three mutations, preferably from one to two and more preferably a conservative mutation. A conservative mutation is for example the replacement of a basic residue with another basic residue, an acid residue with another acid residue or a neutral residue with another neutral residue. The present invention also includes peptides that include up to two additional amino acids at the N or C terminal, preferably selected from those present in the corresponding positions in the tubulin sequence.

These sequences are located on the interaction surface between two adjacent αβ units, that is, precisely in the spatial region involved in the polymerization process. The interaction takes place in a different region from the domains used by previously known anticancer drugs targeting tubulin.

In fact, the peptides object of the invention do not interact with one of the sites already known for tubulin but rather act by blocking the surface of the protein.

Furthermore, the site of interaction of the inhibitors of the invention involves highly conserved amino acids in tubulin as they are essential for its polymerization, and therefore the appearance of drug resistance due to mutations is unlikely.

It follows that the proposed inhibitors can be used in the presence of other inhibitors at reduced doses, thus reducing side effects.

The peptides of the invention, which can be prepared using conventional peptide synthesis techniques, can be formulated in suitable pharmaceutical compositions preferably suitable for parenteral administration, given the peptide nature of the active ingredients, even if other routes of administration such as for example for example the oral route.

The compositions of the invention can be used for the treatment of tumor forms that respond to a treatment capable of inhibiting the polymerization of tubulin. The peptides of the invention may possibly be associated with taxane derivatives (paclitaxel, docetaxel), vincamine, etoposide and derivatives or analogues.

The peptides of the invention may also be useful as antiproliferative, anti-inflammatory agents and for the treatment of dermatological or neuro degenerative infections and diseases

The properties of the peptides of the invention have been highlighted using both computational and experimental methods, as shown below.

Computational methods

Three "all-atoms" molecular dynamics simulations were conducted using the AMBER force field on the systems obtained by "docking" the

peptide 1 and peptide 3 on unit a and peptide 2 on unit β of tubulin.

Then the binding energies of the peptides were calculated using the

technique called MM-GBSA using the structures obtained from the dynamics

molecular. The amount of each residue to the binding energy was evaluated

by performing a "computational alanine scanning". They were also conducted

docking studies.

Computational results peptide 1

The binding energy for peptide 1 amounts to 47% compared to

binding offered by the complete interaction between two pairs of heterodimers.

The most significant interactions of the peptide are reported in Table la

1 with the residues of unit a of tubulin and in Tab. 2a the values obtained

from alanine scanning.

Pairwise Decomposition for Peptide 1

Res Res Eint (kcal / mol)

SER3THR349-0.67

VAL4PRO348-0.87

VAL4THR349-1.53

VAL4PHE3 5i -0.54

SER5THRug-3.89

SER5PHE3 5i -2.19

ASPèPHE351-1.64

ASP6LYS352-13.92

ASP6VAL353-8.07

THR7THRU9-3.16

THR7GLY350-1.78

TEI Ri PHE3 5i -1.34

THR7LYS352-1.80

VAL9LYS353 - 1.99

Table la: Decomposition of the Association Energy for peptide 1 Alanine Scanning of Peptide 1

residue ΔΔ Gpeptidei (kcal / mol)

SERIES 0.74

PRO20.33

LYS3-0,2

VAU 3.73

SER52.67

ASP618.23

THR71.76

VAU -0.01

VAL9-0.66

Table 2a: Alanine Scanning for peptide 1 The results show that peptide 1 interacts mainly with the residues of unit a. More specifically, peptide 1 is able to establish an electrostatic interaction due to a salt bridge between Asp6 and Lys352 and a series of hydrophobic interactions involving Val4, Thr7e Val9 with Thr349, Phe351 and Lys352. The geometric analysis of the conformations obtained during the molecular dynamics also show the establishment of hydrogen bridging bonds between the Asp6-Phe351, Asp6-Val353 and Thr7-Thr349 pairs.

During the simulation, the aforementioned interactions remain stable, thus confirming that peptide 1 is able to join the β unit of tubulin, right on the surface that should be attacked by other heterodimers, thus influencing the polymerization process.

The conformations obtained from the molecular dynamics were subjected for further confirmation to a "docking" procedure which confirmed the binding of the peptide with an interaction energy of approximately 13.0 Kcal / mol.

Computational results peptide 2 (Xaa = Cys)

The binding energy for peptide 2 amounts to 30% compared to

binding offered by the complete interaction between two pairs of heterodimers. THE

results for peptide 2 are as we will see specularly similar with respect to

those of peptide 1, as they are located in positions facing the

interproteic surface of tubulin.

The most significant interactions of the peptide are reported in Table 1b

2 with the residues of the β unit of tubulin and the values obtained in Table 2b

daH alanine scanning.

Pairwise Decomposition for Peptide 2

Res Res Eint (kcal / mol)

THR5VAL, 1485-1.28

THR5SER 1486 -2.58

THR5ASP 1487 -1.24

THR5THR, 1 488-3.07

THR5V AL1489 -1.00

ΡΗΕη VAL, 1485 -0.71

ΡΗΕη SER, 1486 -2.95

ΡΗΕη ASP1487 -1.46

ΡΗΕη THR, 1488 -0.31

LYS9ASP 1489 -7.31

LYS9THR, 1488 -0.71

VALID THR, 484 -0.82

VAL, or SER 1486 -0.61

Table lb: Decomposition of the Association Energy for peptide 2 Alanine Scanning of Peptide 2

residue ΔΔ Gpeptidel (kcal / mol)

TRP20.22

CYS3-0.10

PRO42.32

THR53.66

ΡΗΕη 0.67

LYSs 3.26

VAL95.83

Table 2b: Alanine Scanning for Peptide 2

The results show that peptide 2 mainly interacts with the residues of the β unit which constitute the subsequence from which peptide 1 is derived. More specifically, peptide 2 is also able to establish an electrostatic interaction due to a salt bridge between rAsp1487 and Lys8, and a series of hydrophobic interactions involving the same amino acids already highlighted for peptide 1.

During the simulation, the aforementioned interactions remain stable, thus confirming that peptide 2 is able to join the β unit of tubulin, right on the surface that should be attacked by other heterodimers, thus influencing the polymerization process.

The conformations obtained from the molecular dynamics were subjected for further confirmation to a docking procedure which confirmed the binding of the polypeptide with an interaction energy equal to about 16.0 Kcal / mol.

Computational results peptide 3

The binding energy for peptide 3 amounts to 40% compared to the binding offered by the complete interaction between two pairs of heterodimers. Table shows the most significant interactions of peptide 3 with the residues of unit a and table 2c shows the results of alanine scanning.

Table le: Decomposition of the Association Energy for the peptide 3

Table 2c: Alanine Scanning for Peptide 3

The results show that peptide 3 interacts with various residues of the a subunit, and in particular it establishes an electrostatic interaction due to the contact between Lys4 and Asp438, hydrogen bonds between Trp9e Val26o and between Arg3e Ser439. There are also hydrophobic interactions between Phe6e Val260, between His8e Tyr262, and between Trp9, Val260, and Tyr262.

During the simulation, the aforementioned interactions remain stable, thus confirming that the peptide 3 is able to join the unit a of the tubulin, right on the surface that should be subject to attack by other heterodimers, thus influencing the polymerization process.

The conformations obtained from the molecular dynamics were subjected for further confirmation to a docking procedure which confirmed the binding of the polypeptide with an interaction energy equal to about 16 Kcal / mol.

Experimental methods

The efficacy of peptide 1 was also evaluated in a tubulin polymerization test conducted in the presence and absence of the peptide.

The test consists in monitoring the absorbance of a tubulin solution over time. The polymerization of tubulin generates microtubules, the presence of which increases the absorbance over time. The inhibition of polymerization manifests itself as a reduction in absorbance compared to the similar test conducted in the absence of the peptide.

The effect of the presence of the peptide is manifested not only by a reduction in absorbance over time but also by a marked difference in the initial polymerization rates, highlighted by the different initial slope of the curves shown in the Figure.

Claims (7)

CLAIMS 1. A peptide having one of the following sequences: - Ser-Pro-Lys-Val-Ser-Asp-Thr-Val-Val; - Ala-Trp-Xaa-Pro-Thr-Gly-Phe-Lys-Val-Gly (Xaa is Cys or Ala); - Phe-Arg-Arg-Lys-Ala-Phe-Leu-His-Trp-Tyr-Thr-Gly, their analogs or derivatives. 2. The peptide of claim 1 which is Ser-Pro-Lys-Val-Ser-Asp-Thr-Val-Val. 3. The peptide of claim 1 which is Ala-Trp-Xaa-Pro-Thr-Gly-Phe-Lys-Val-Gly (Xaa is Cys or Ala). 4. The peptide of claim 1 which is Phe-Arg-Arg-Lys-Ala-Phe-Leu-His-Trp-Tyr-Thr-Gly. 5. Pharmaceutical compositions comprising as active ingredients the peptides of claims 1-4. 6. Use of the peptides of claims 1-4 for the preparation of anti-tumor medicaments. 7. Use of the peptides of claims 1-4 for the preparation of antiproliferative, anti-inflammatory medicaments and for the treatment of dermatological and neuro degenerative infections and diseases. Milan, 13 December 2007