METHOD OF CONSTRUCTING PROTAC BY USING DOUBLE TARGETS

Abstract

The present invention provides a method for constructing PROTAC by using double targets, and a specific construction method and application of a double-target. PROTAC constructed by using the method. The present invention proposes for the first time the concept of PROTAC with double-target design, namely, the Target protein I is degraded by PROTAC when a specific E3 is selected, meanwhile, the Target protein II is increased for the degradation of the E3 natural substrate protein is hindered due to the competition of PROTAC to E3. By using this method, the present invention constructs for the first time a double-target PROTAC which is capable of not only degrading Smad3 via target ubiquitination but also simultaneously up-regulating the HIF-α protein level, which theoretically plays the role of renal protection, such as anti-fibrosis and the treatment of renal anemia, via multi channels.

Claims

1. A method of constructing PROTAC by using double targets, wherein including steps of: (1) using harmful intracellular protein as a first target protein, and screening a small molecular compound specifically bind to the first target protein to serve as a recognition ligand of the first target protein; (2) selecting a protective protein within cells naturally expressing the first target protein to serve as a second target protein, using a specific E3 ligase of the second target protein as E3 of PROTAC recognition, and screening and determining a small molecular compound capable of specifically binding to E3 ubiquitin ligase to serve as a recognition ligand of the E3 ubiquitin ligase; (3) screening a compound capable of stably binding to both the recognition ligand of the first target protein obtained in Step (1) and the recognition ligand of E3 ubiquitin ligase obtained in Step (2) to serve as Linker; and (4) connecting the recognition ligand of the first target protein obtained in Step (1) and the recognition ligand of E3 ubiquitin ligase obtained in Step (2) by using the Linker obtained in Step (3) so as to obtain the double-target PROTAC.

2. The method according to claim 1, wherein the first target protein is Smad3, and the recognition ligand of the first target protein is a compound expressed by Formula (I), ##STR00008##

3. The method according to claim 1, wherein the second target protein is HIF-α protein.

4. The method according to claim 3, wherein the E3 ubiquitin ligase is VHL E3 ubiquitin ligase.

5. The method according to claim 1, wherein the recognition ligand of the E3 ubiquitin ligase is a compound expressed by Formula (II), ##STR00009##

6. The method according to claim 1, wherein the Linker is butanedioic acid.

7. The method according to claim 1, wherein the double-target PROTAC is a compound expressed by Formula (III), ##STR00010## wherein: R.sub.1 is hydrogen, straight-chain alkyl or substituted straight-chain alkyl, cycloalkyl, alkenyl or substituted alkenyl, carbonyl or substituted carbonyl, five-membered or six-membered heterocycle, benzene ring or benzene ring containing substituent; R.sub.2 is hydrogen, straight-chain alkyl or substituted straight-chain alkyl, cycloalkyl, carbonyl or substituted carbonyl, five-membered or six-membered heterocycle, five-membered aromatic heterocycle or five-membered aromatic heterocycle containing substituent, benzene ring or benzene ring containing substituent, six-membered aromatic heterocycle or six-membered aromatic heterocycle containing substituent; and A is carbon atom or nitrogen atom.

8. The method according to claim 7, wherein R.sub.1 is hydrogen, straight-chain alkyl or substituted straight-chain alkyl; R.sub.2 is hydrogen, straight-chain alkyl or substituted straight-chain alkyl, cycloalkyl, five-membered or six-membered heterocycle, five-membered aromatic heterocycle or five-membered aromatic heterocycle containing substituent, benzene ring or benzene ring containing substituent, six-membered aromatic heterocycle or six-membered aromatic heterocycle containing substituent; and A is carbon atom or nitrogen atom.

9. The method according to claim 8, wherein R.sub.1 is hydrogen, methyl, ethyl, isopropyl; R.sub.2 is hydrogen, methyl, ethyl, isopropyl, morpholine, piperazine, thiophene or thiophene ring containing substituent, thiazole or thiazole ring containing substituent, benzene ring or benzene ring containing substituent, pyridine ring or pyridine ring containing substituent; and A is carbon atom or nitrogen atom.

10. The method according to claim 9, wherein the structural formula of the PROTAC is one of the structural formulae below: ##STR00011##

11. A double-target PROTAC constructed by using the method according to claim 1.

12. A drug for anti-fibrosis, treatment of renal anemia, diabetes, diabetic nephropathy and/or protection of renal function comprising the double-target PROTAC according to claim 11.

13. The drug according to claim 2, wherein the fibrosis is renal fibrosis.

Description

DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a diagram of target protein degradation mediated by PROTAC.

[0031] FIG. 2 is a mode pattern of Smad3/HIF-α double-target PROTAC construction mechanism. Note: SMC: small molecular compound.

[0032] FIG. 3 is a mode pattern of research hypothesis.

[0033] FIG. 4 is a combination mode pattern of Smad3 target protein ligand and Smad3 protein in Example 1 of the present invention.

[0034] FIGS. 5(A), 5(B), 5(C) and 5(D) are diagrams showing the structure identification (MS and HPLC) of the double-target PROTAC in Example 1 of the present, invention. Note: FIGS. 5(A) and 5(B) are respectively MS diagrams of butanedioic acid and PEG; FIGS. 5(C) and 5(D) are respectively HPLC diagrams of butanedioic acid and PEG.

[0035] FIGS. 6(A) and 6(B) are diagrams showing the activity comparison of double-target PROTAC with different Linkers in Example 1 of the present invention (Western blot). Note: comparison with TGF-β1 control group, *P<0.05, **P<0.01.

[0036] FIG. 7 is a diagram showing the pharmacological effects of Smad3/HIF-α double-target PROTAC on renal fibroblasts in Example 1 of the present invention.

EMBODIMENTS

[0037] In order to better explain the purpose, the technical solution and the advantages of the present invention, the present invention will be further elaborated with reference to the specific example.

[0038] Based on our research basis for constructing a single-target PROTAC, the present invention proposes for the first time to construct a Smad3/HIF-α double-target. PROTAC, and the construction mechanism is shown in FIG. 2.

[0039] In our anticipation, the combination of the different anti-fibrosis mechanism effects of two targets can not only significantly enhance the renal protection of PROTAC, but also complement with each other, i.e. to overcome, by means of Smad3 degradation, the pro-fibrosis effect that HIF-1α may have in the early stage of renal injury while preserving the powerful renal protection of HIF-2α.

[0040] In summary, we put forward the following research hypothesis: in the progress of CKD, due to the excessive activation of Smad3 signal and relative insufficiency of HIF-α, the application of Smad3/HIF-α double-target PROTAC can block the fibrosis of the Smad3 signal pathway, as well as has the function of HIF-α stabilization, thereby achieving multiple goals of double targets, inhibiting renal fibrosis, renal function protection, and renal anemia prevention and treatment through multiple pathways, see FIG. 3.

[0041] Based on our preliminary research basis of screening specifically recognized Smad3 protein small molecules as the target protein recognition ligand, small molecules specifically bind to VHL are used as the E3 recognition ligand to construct PROTAC, and Smad3/HIF-α double-target PROTAC is constructed and synthesized, which can significantly degrade intercellular Smad3 protein of rat kidney fibroblasts while up-regulating the protein level of HIF-2a.

Example 1

[0042] I. Seek Small Molecules Specifically Bind to Target Protein Smad3 by Combining Computer Virtual Screening Technology and SPR Technology

[0043] Based on our previous research results, we have successfully screened out a small molecule that can specifically bind to the target protein Smad3, the structural formula is shown in Formula (I), and it is confirmed that PROTAC constructed by using this small molecule as the target protein ligand can degrade Smad3 protein via target ubiquitination, as a result, for the double-target PROTAC newly constructed this time, we still use this small molecule as the target protein ligand.

##STR00005##

[0044] II. Determination of the Binding Site of Target Protein Small Molecule Ligand with the Linker

[0045] Molecule docking is performed for the previously screened small molecules and Smad3, see FIG. 4. It can be seen that the amine group (—NH.sub.2) on the upper part of No. 8 small molecule forms a hydrogen bond with the carboxyl group at the end of Glu-245 residue of Smad3, N on the pyridine ring of the molecule forms a hydrogen bond with —NH on the main chain of the residue His-248, and the dihydrogen bond holds the small molecule here; secondly, the pyridine ring on the right part of the small molecule forms a π-π interaction with the benzene ring at the end of the Phe-247 residue, and the benzofuran ring on the left part and the benzene ring at the end of the Phe-268 residue also forms a π-π interaction. From the interaction diagram, it can be seen that the benzofuran ring and pyridine ring of the small molecule extend into the interior of the protein and adapt to the active pocket of the protein, while the amino group at the upper part faces the exterior space of the protein, so the modification of the amino group has little effect on the binding activity of No. 8 small molecule and the Smad3 protein, and can serve as a site connecting with Linker. In addition, the amino group is an active group for chemical reaction, and its link with Linker can be easily realized.

[0046] III. Determination of the Binding Site of VHL E3 Recognition Ligand Small Molecule with the Linker

[0047] Small molecule specifically bind to VHL is adopted in this invention as VHL E3 recognition ligand, and the structural formula is shown by Formula (II),

##STR00006##

[0048] IV. Construction of Double-Target PROTAC with Different Linkers

[0049] Under the premise of non-toxicity, good water solubility and small molecular weight, the present invention initially selects two small molecules, including a long one (polyethylene glycol, PEG) and a short one (butanedioic acid) as Linkers for constructing double-target PROTAC: PROTAC-PEG (see Compound 7) and PROTAC-butanedioic acid (see Compound 1).

##STR00007##

[0050] V. Structural Verification of PROTAC with Different. Linkers

[0051] The structure of PROTAC is analyzed to be correct by means of mass spectrometry (MS) and high performance liquid chromatography (HPLC), see FIGS. 5(A), 5(B), 5(C) and 5(D).

[0052] VI. Activity Comparison of Double-Target PROTAC Constructed by Different Linkers

[0053] Rat kidney fibroblasts (NRK49F cell line) are cultured in vitro, passage 1:3 or 1:4 to six-well plates before cell fusion, and are divided into a TGF-β1 control group (TGF-β1 10 ng/ml), a PROTAC-PEG action group (TGF-β1 10 ng/ml+PROTAC-PEG of different concentrations: 1, 5, 25, 125 nM), and a PROTAC-butanedioic acid action group (TGF-β1 10 ng/ml+PROTAC-butanedioic acid of different concentrations: 1, 5, 25, 125 nM); the cells are lysed after 48 hours of culture and are used for Western blot. The results show that PROTAC-butanedioic acid degrades the target protein Smad3 in a concentration-dependent manner, its activity is significantly higher than that of PROTAC-PEG, and its effective concentration is as low as 25 nM, see FIGS. 6(A) and 6(B).

[0054] By means of experiments in vitro, it has been confirmed that the PROTAC can degrade intracellular Smad3 in a concentration-dependent manner, and at the same time significantly up-regulate the protein level of HIF-2a, and the effective concentration is lower than 25 nM, see FIG. 7.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to preferable example, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the substance and scope of the technical solution of the present invention.