CN111393519A - As KRASG12CNovel stapled peptides of/SOS 1 inhibitor and uses thereof - Google Patents

Abstract

The invention discloses a KRAS^G12CAlso provided are compositions comprising these stapled peptides which are to some extent more stable in their α -helix conformation, and methods of using such peptides in the treatment of cancer, which stapled peptides are more stable with KRAS^G12CThe stapled peptides of the invention have greatly enhanced plasma stability, are derived from the α -helix binding region of SOS1 protein and KRAS protein, inhibit activation of the KRAS protein, the preparation method thereof and the medicaments containing the stapled peptidesCompositions, and their use, alone or in combination with other compounds, for the prevention or treatment of cancer (e.g., non-small cell lung cancer).

Description

As KRASG12CNovel stapled peptides of/SOS 1 inhibitor and uses thereof

Technical Field

The present invention relates to a stapled peptide derived from the α -helix binding region of the SOS1 protein and the KRAS protein.

Background

Lung cancer is the leading cause of cancer death worldwide (Cheng and Planken 2018), severely harming human life and health, and over the past decades treatment of lung cancer has received great attention (Wang, Huang et al 2019). non-small cell lung cancer (NSC L C) accounts for 80% of all lung cancer cases (Jemal, Bray et al 2011), Ras proto-oncogenes are the most common mutant genes in NSC L C (Prior, L ewis et al 20152012, L i, L iu et al 2018), mutations are detected in about 25% of all tumors (de Castro Carpeno and Belda-Iniesta 2013), v-Ras-2 Kirsten rat sarcoma virus (KRAS) accounts for 90% of lung adenocarcinoma Ras mutations (John C.

RAS proteins have the potential to transform cells when point mutations occur at amino acids 12,13 or 61 in the RAS gene, 97% of KRAS mutations in NSC L C involve amino acids 12 and 13, with mutations at amino acid 12 accounting for about 80% of the GAP proteins stimulating KRAS, mutations at this position slowing down GTP to GDP and increasing GTP levels due to GAP acting as a catalyst to accelerate gtpase activity RAS proteins function as molecular switches that cycle between GDP-bound inactive and GTP-bound active states and mediate this cycle by Gtpase Activating Proteins (GAP) and guanine nucleotide exchange factors (GEF), where SOS1 and SOS 2 are members of GEF (borinfi, KAR L et al 1992, rick-Sjodin, margagen 1998, Bos, yak et al, sec, yak ph 52, sec.

Compounds currently investigated to inhibit KRAS activity include destruction effectors such as guanine nucleotide exchange factors and against KRAS^G12CThe compound of (5), and the like, and further having a polypeptide secondary structure as a supporting skeleton in protein-protein interactionThe polypeptide fragment is not stable to form a secondary structure required for binding after leaving the whole protein structure, is easy to form a random coil conformation so as to cause the reduction of the binding activity, is more easily degraded by peptidase, and cannot be directly used as a medicine₂X represents (S) - α -methyl- α -pentenylglycine (S5) (L eshchiner, Parkhitko et al 2015), but for the development of polypeptide drugs, too long a sequence often causes problems of reduced solubility, poor stability, increased production cost and the like, and thus, most of the polypeptide drugs on the market have a length of about 10 amino acids.

Despite the deep understanding of the mechanisms of KRAS and its pathological mutations, the study of effective stable targeted drugs against KRAS mutations is still remote and remains a formidable challenge.

Disclosure of Invention

The present invention relates to stapled peptides derived from the α -helix binding region of the SOS1 protein and the KRAS protein, which inhibit activation of the KRAS protein, their preparation, pharmaceutical compositions containing them, and their use, alone or in combination with other compounds, for the prevention or treatment of cancer, such as non-small cell lung cancer, and to their use.

The technical scheme of the invention is as follows: a stapled peptide comprising the amino acid sequence A₀B₀C₀D₀E₀F₀G₀A₁B₁C₁D₁Wherein: a. the₀Is F or a conservative substitution; b is₀Is G or a conservative substitution; c₀Is I or a conservative substitution; d₀Is Y or a conservative substitution; e₀L or a conservative substitution F₀Is T or a conservative substitution; g₀Is N or a conservative substitution; a. the₁Is I or a conservative substitution; b is₁L or a conservative substitution C₁Is K or conservativeSubstitution; d₁Is T or a conservative substitution;

the stapled peptide comprising an α -helix and two unnatural amino acids X cross-linked at the i and i +3 positions, or at the i and i +4 positions, or at the i and i +7 positions by a macrocycle-forming linker;

the stapled peptide is selected from Ac-FGIX L TNX L KT-NH₂(SEQ ID NO.1)；Ac-FFGIXLTNXLKTE-NH₂(SEQ ID NO. 2); wherein two unnatural amino acids X are end-capped with an olefin and cross-linked through a macrocycle-forming linker;

wherein the amino acid sequence of SEQ ID NO.1 is phenylalanine-glycine-isoleucine-unnatural amino acid X-leucine-threonine-asparagine-unnatural amino acid X-leucine-lysine-threonine.

The amino acid sequence of SEQ ID No.2 is phenylalanine-glycine-isoleucine-unnatural amino acid X-leucine-threonine-asparagine-unnatural amino acid X-leucine-lysine-threonine-glutamic acid.

The 20 common naturally occurring α -amino acids and their three-letter one-character designations are as follows:

alanine (Ala or a), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (L eu or L), lysine (L ys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Further, the two unnatural amino acids X are (S) - α -methyl- α -pentenylglycine (S5).

Further, the amino acid sequence is Ac-FGIX L TNX L KT-NH₂。

Further, the two unnatural amino acids X are crosslinked at the i and i +4 positions by a macrocycle-forming linker.

Further, the amino acid sequence is 11 amino acids in length; and the position of the unnatural amino acid is fixed.

A pharmaceutical composition comprising a stapled peptide as described above.

Further, the pharmaceutical composition comprises a drug for treating cancer.

A method of treating cancer, comprising administering to a patient in need thereof a stapled peptide as described above.

Further, the cancer is KRAS^G12CA cancer of interest; such as pancreatic cancer, lung cancer, colorectal cancer, etc.; wherein the cancer is non-small cell lung cancer; the patient with non-small cell lung cancer carries mutant KRAS^G12CA protein.

Further, the stapled peptide has selective inhibitory effect on H358 cells;

the stapled peptides have anti-proteolytic ability and plasma stability.

The stapled peptides according to the present invention include covalent bonds between two amino acid side chains in the peptide, and the stapling of the polypeptide can be used to physically confine the peptide to a particular conformation (e.g., physically confine the peptide to its native α -helix state), thereby enhancing the pharmacological properties of the peptide by helping to retain the native structure required for interaction with a target molecule, increasing cell permeability, and/or protecting the peptide from proteolytic degradation.

A stapled Peptide according to the present invention is designed based on the inhibition of the KRAS/SOS1 protein interaction, the RAS protein acting as a molecular switch, cycling between a GDP-bound inactive state and a GTP-bound active state, mediated by the GTPase Activating Protein (GAP) and the guanine nucleotide exchange factor (GEF), wherein none of the heptans 1 and 2(SOS1 and 2) are members of the guanine nucleotide exchange factor (GEF), the SOS activating the RAS by inserting a SOS helix hairpin directly into the RAS switch or the water-mediated interaction between RAS and guanine nucleotides, the strategy being adopted to extract the α -helix Peptide alone in the folding subdomain of the protein-protein interaction with the polypeptide secondary structure as the supporting backbone, to construct an active polypeptide drug precursor that has the potential to act selectively on the KRAS protein by means of chemical synthesis, to develop a stapled Peptide, to mimic the SOS1, to form a full-carbon backbone by applying the sidechain structure to form a loop, to synthesize the SADB-18-loop Peptide, thus further shorten the conformation of the synthesized SAAS-18-GDH-Peptide, to the polypeptide sequence, to further shorten the length of the stapled polypeptide.

The sequence is defined below:

Peptide-DB-1：Ac-FGIXLTNXLKT-NH₂(SEQ ID NO:1)；

Peptide-DB-2：Ac-FFGIXLTNXLKTE-NH₂(SEQ ID NO:2)；

among these, two peptides were chosen to describe their activity: different lengths of stapled peptides Peptide-DB-1 and Peptide-DB-2; in biological experiments, the stapled peptide SAH-SOS1A was synthesized to serve as a positive control stapled peptide.

The sequences are listed in table 1 below:

table 1:

SEQ ID NO.	name (R)	Sequence of
			SEQ ID NO:1	Peptide-DB-1	Ac-FGI S5LTN S5LKT-NH2
SEQ ID NO:2	Peptide-DB-2	Ac-FFGI S5LTN S5LKTE-NH2
			SEQ ID NO:3	SAH-SOS1A	Ac-RRFFGIS5LTNS5LKTEEGN-NH2(existing)

Is denoted as "S₅"amino acid (S)" means (S) - α -methyl- α -pentenylglycine linked by an all carbon i to i +4 crosslink linker containing one double bond;

"Ac" represents acetyl.

These stapled peptides are also represented in table 2a and table 2b below:

table 2 a:

table 2 b:

when using stapled peptides to physically confine the peptide to its native α -helix state, it is desirable to form a staple between the i and i +3 positions of the peptide, or between the i and i +4 positions of the peptide, or between the i and i +7 positions of the peptide, because the amino acid side chains at the i, i +3, i +4, and i +7 positions will be on the same face of the helix when the α helix is formed.

Stapled peptides according to the invention form staples between the i and i +4 positions, and an example of stapled peptides with staples between the i and i +4 positions is shown in fig. 1.

The preparation method of the staple peptide comprises the following steps:

methods of synthesizing the compounds described herein are known in the art; however, the following exemplary method was used; it will be appreciated that the various steps may be performed in an alternative order or sequence to obtain the desired compound; synthetic chemical transformations and protecting group methods (protection and deprotection) useful in the synthesis of the compounds described herein are known in the art.

The peptides of the invention may be prepared by chemical synthesis methods, which are well known to the skilled person; one way to prepare the peptides described herein is to use Solid Phase Peptide Synthesis (SPPS); the C-terminal amino acid is linked to the cross-linked polystyrene resin through an acid-labile bond with a linker molecule; the resin is insoluble in a solvent used for synthesis, so that redundant reagents and byproducts can be washed away relatively simply and quickly; the N-terminus is protected by an Fmoc group, which is stable in acid but removable by base; any side chain functional groups are protected by base stable acid labile groups. The N-terminus of the synthetic peptide was acetylated, while the C-terminus was aminated.

After the unnatural amino acid S5 is incorporated into the precursor polypeptide, the terminal olefin is reacted with a metathesis catalyst, resulting in the formation of a stapled peptide.

In such embodiments, the macrocyclization reagent or the macrocycle-forming reagent is a metathesis catalyst, including but not limited to a stable late transition metal carbene complexation catalyst, such as a group VIII transition metal carbene catalyst; for example, such catalysts have Ru and Os as metal centers, a +2 oxidation state, a number of electrons of 16, and are penta-coordinated.

In some embodiments, the contacting step is carried out in a solvent selected from the group consisting of protic solvents, aqueous solvents, organic solvents, and mixtures thereof; for example, the solvent may be selected from H₂O、THF、THF/H₂O、tBuOH/H2O、DMF、DIEA、CH₃CN or CH₂Cl₂、ClCH₂CH₂Cl or a mixture thereof; in a specific embodiment, DMF is used.

The peptide was purified and characterized by standard methods.

In a particular embodiment, the method for preparing the stapled peptides according to the invention comprises at least the following steps:

providing a plurality of peptides comprising a protecting group, each peptide being immobilized on a solid support; exposing a deprotection reagent to the immobilized peptide to remove the protecting group from at least a portion of the immobilized peptide; removing at least a portion of the deprotecting reagent; dissolving the protected amino acid residue in a solvent, preferably DMF; using a coupling agent, preferably HATU; using an alkaline agent, preferably DIEA; exposing the protected amino acid residue and the coupling reagent to the immobilized peptide such that at least a portion of the activated amino acid residue binds to the immobilized peptide to form a newly bound amino acid residue; and removing at least part of the activated amino acid residues not bound to the immobilized peptide; exposing the final linear polypeptide to a Grubb's catalytic reagent for a displacement reaction to produce stapled peptide; exposing the final stapled peptide to a cleaving agent for final deprotection; the final stapled peptide is precipitated, purified and lyophilized, preferably to a purity of greater than 90%.

The invention has the advantages that the stapled Peptide-DB-1 has α -helix content higher than that of Elizaveta S, L eshchinra and other synthesized SAH-SOS1A in water environment or membrane simulation environment, and the stapled Peptide-DB-1 with shortened amino acid sequence can not only maintain α -helix content of the original stapled Peptide, but also has α -helix conformation which is more stable to a certain extent (figure 2).

The stapled Peptide of the present invention, Peptide DB-1, compares to SAH-SOS1A designed and synthesized by Elizaveta S. L eshchinera et al, which is a Peptide of the present invention, with KRAS^G12CThe protein has higher affinity SAH-SOS1A with KRAS^G12CThe affinity of the protein is 46.9 mu M, and the Peptide-DB-1 and KRAS^G12CThe affinity of the protein can reach 5.05 mu M, and the affinity is improved by nearly 10 times.

The stapled Peptide-DB-1 has selective inhibition effect on H358 cells.

The stapler Peptide-DB-1 has better capability of resisting the enzymolysis of trypsin and α -chymotrypsin, 6 percent of Peptide-DB-1 is not degraded after being incubated for 24 hours in the trypsin, and 70 percent of Peptide-DB-1 is not degraded after being incubated for 24 hours in α -chymotrypsin.

Compared with the SAH-SOS1A designed and synthesized by Elizaveta S. L eshchinera et al, the stapled Peptide-DB-1 has greatly enhanced plasma stability, no characteristic peak is observed when the SAH-SOS1A is incubated for 0 minute after the plasma is added, and the Peptide-DB-1 is still stable after being incubated for 24 hours in rat plasma.

Drawings

FIG. 1: a schematic of the structure of the stapled peptides of the invention;

FIG. 2A: the CD spectrum structure diagram of Peptide-DB-1 in deionized water in the range of 190-260 nm;

FIG. 2B: the CD spectrum structure diagram of Peptide-DB-2 in deionized water in the range of 190-260 nm;

FIG. 2C: the CD spectrum structure diagram of the SAH-SOS1A in the deionized water in the range of 190-260 nm;

FIG. 2D: the CD spectrum structure diagram of Peptide-DB-1 in PBS (50% TFE) in the range of 190-260nm in the invention;

FIG. 2E: the CD spectrum structure diagram of Peptide-DB-2 in PBS (50% TFE) in the range of 190-260nm in the invention;

FIG. 2F: the CD spectrum structure of SAH-SOS1A in PBS (50% TFE) in the invention is in the range of 190-260 nm;

FIG. 3A: SAH-SOS1A and KRAS in the present invention^G12CGraph of protein dose-dependent binding and dissociation curves;

FIG. 3B: in the invention, Peptide-DB-1 and KRAS^G12CGraph of protein dose-dependent binding and dissociation curves;

FIG. 3C KRAS determined by B L I in the present invention^G12CKinetic structure diagram of the interaction with stapled peptides;

FIG. 4A: the structure of the cell viability of SAH-SOS1A and Peptide-DB-1 of the invention after 24 hours incubation in H358 cells;

FIG. 4B: cell viability profile of SAH-SOS1A and Peptide-DB-1 of the invention after 24 hours incubation in A549 cells;

FIG. 5A: in the invention, the SAH-SOS1A inhibits KRAS^G12CStructure of association with nucleotides;

FIG. 5B: in the invention, Peptide-DB-1 inhibits KRAS^G12CStructure of association with nucleotides;

FIG. 6A: the structure diagram of cell cycle distribution analysis is carried out by flow cytometry under the influence of Peptide-DB-1 on a KRAS mutant cell line in the cell cycle process;

FIG. 6B: the structure diagram of the cell distribution bar of the H358 cells in different stages of the cell cycle in the invention;

FIG. 6C: the structure chart of cell distribution bars of A549 cells in different stages of the cell cycle in the invention;

FIG. 6D: structural diagram of the western blot analysis of related cyclins in H358 cells of the invention;

FIG. 6E: a structural diagram of western blot analysis of related cyclins in a549 cells of the invention;

FIG. 7A: in the invention, Peptide-DB-1 induces H358 and A549 cell apoptosis by flow cytometry; q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, Q4 represents normal cells; the sum of Q2 and Q3 represents a structural map of the apoptosis rate;

FIG. 7B: a graph of the percentage of apoptotic cells in H358 cells of the invention;

FIG. 7C: a graph of the percentage of apoptotic cells in a549 cells in the invention;

FIG. 7D: structural diagram of western blot analysis of related apoptotic proteins in H358 cells of the invention;

FIG. 7E: the structure of the western blot analysis of the relevant apoptotic proteins in a549 cells of this invention;

FIG. 8A is a diagram showing the chromatographic structure of HP L C of Peptide-DB-1 after 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr and 24 hr of trypsin degradation in the present invention;

FIG. 8B is a diagram showing the chromatographic structure of HP L C of Peptide-DB-1 in the present invention after degradation of α -chymotrypsin for 0 min, 30 min, 1 h, 2h, 6 h, 12h and 24 h;

FIG. 8C: map of the percentage of Peptide-DB-1 and hydrolysate at different time points for trypsin in the present invention;

FIG. 8D is a graph showing the percentage of Peptide-DB-1 and hydrolysates at various time points for α -chymotrypsin in accordance with the invention;

FIG. 8E: the present invention was performed at 37 ℃ at various time points in NH containing trypsin₄HCO₃Map of the percentage of Peptide-DB-1 in buffer;

FIG. 8F shows NH containing α -chymotrypsin at various time points at 37 ℃ in accordance with the invention₄HCO₃Map of the percentage of Peptide-DB-1 in buffer;

FIG. 9A: chromatograms of SAH-SOS1A and Peptide-DB-1 in the present invention with acetonitrile-water (1: 1; containing 0.1% formic acid) as solvent;

FIG. 9B: blank plasma chromatograms in the invention;

FIG. 9C is a chromatogram of SAH-SOS1A of 1300. mu.g/m L of the present invention incubated for 0 min in plasma;

FIG. 9D is a chromatogram of 500. mu.g/m L of Peptide-DB-1 in plasma incubated for 0 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr, respectively;

FIG. 9E: schematic representation of SAH-SOS1A and Peptide-DB-1 in the present invention;

FIG. 9F: a structural diagram showing the change in peak area of Peptide-DB-1 in 24 hours in the present invention.

Detailed Description

As shown in the figure, a stapled peptide, the stapled peptide comprises an amino acid sequence A₀B₀C₀D₀E₀F₀G₀A₁B₁C₁D₁Wherein: a. the₀Is F or a conservative substitution; b is₀Is G or a conservative substitution; c₀Is I or a conservative substitution; d₀Is Y or a conservative substitution; e₀L or a conservative substitution F₀Is T or a conservative substitution; g₀Is N or a conservative substitution; a. the₁Is I or a conservative substitution; b is₁L or a conservative substitution C₁Is K or a conservative substitution; d₁Is T or a conservative substitution;

the stapled peptide comprising an α -helix and two unnatural amino acids X cross-linked at the i and i +3 positions, or at the i and i +4 positions, or at the i and i +7 positions by a macrocycle-forming linker;

the stapled peptide is selected from Ac-FGIX L TNX L KT-NH₂(SEQ ID NO.1)；Ac-FFGIXLTNXLKTE-NH₂(SEQ ID NO. 2); wherein two unnatural amino acids X are end-capped with an olefin and cross-linked through a macrocycle-forming linker;

wherein the amino acid sequence of SEQ ID NO.1 is phenylalanine-glycine-isoleucine-unnatural amino acid X-leucine-threonine-asparagine-unnatural amino acid X-leucine-lysine-threonine;

the amino acid sequence of SEQ ID No.2 is phenylalanine-glycine-isoleucine-unnatural amino acid X-leucine-threonine-asparagine-unnatural amino acid X-leucine-lysine-threonine-glutamic acid;

the 20 common naturally occurring α -amino acids and their three-letter one-character designations are as follows:

alanine (Ala or a), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (L eu or L), lysine (L ys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Further, the two unnatural amino acids X are (S) - α -methyl- α -pentenylglycine (S5).

Further, the amino acid sequence is Ac-FGIX L TNX L KT-NH₂。

Further, the two unnatural amino acids X are crosslinked at the i and i +4 positions by a macrocycle-forming linker.

Further, the amino acid sequence is 11 amino acids in length; and the position of the unnatural amino acid is fixed.

A pharmaceutical composition comprising a stapled peptide as described above.

Further, the pharmaceutical composition comprises a drug for treating cancer.

A method of treating cancer, comprising administering to a patient in need thereof a stapled peptide as described above.

Further wherein the cancer is KRAS^G12CRelated cancers such as pancreatic cancer, lung cancer, colorectal cancer, and the like; wherein the cancer is non-small cell lung cancer; the patient with non-small cell lung cancer carries mutant KRAS^G12CA protein.

Further, the stapled peptide has selective inhibitory effect on H358 cells;

the stapled peptides have anti-proteolytic ability and plasma stability.

Example 1: preparation and characterization of stapled peptides:

1.1 Synthesis of stapled peptides

Synthesizing the stapling peptide artificially by an SPPS method; the synthesis scale was 0.1mmol, 40-RAM amphiphilic Rink amide resin was loaded at 0.4mmol/g, 5 equivalents excess for protected amino acid; all protected amino acids and the coupling agent HATU were pre-dissolved in DMF to prepare stock solutions at a concentration of 0.5M; the deprotection of the Fmoc group was carried out by swelling the resin in 6ml DMF for 15 min with vortex stirring, after removal of the DMF by filtration, by adding 20% piperidine/DMF solution (6ml) and vortexing for 1 min; the steps are carried out twice; the resin was then washed 3 times with DMF and the desired amino acid (1ml, 0.5mmol), N-Diisopropylethylamine (DIEA) (0.164ml, 1mmol) and HATU coupling agent (1ml, 0.5mmol) were added sequentially and stirred for 5 minutes with vortex stirring and this was done twice by DMF washing between couplings;

for the unnatural amino acid S5, a simple coupling was performed with a stirring time of 1 hour; repeating the deprotection of Fmoc and the coupling of amino acids until the desired linear sequence is obtained; once the linear peptide was synthesized, the displacement reaction was started by vortexing in DCE (6ml) for 2 hours using a first generation Grubbs catalyst (0.04mmol), and between each displacement reaction, the reaction was performed twice with DCM washes.

Once the peptide is stapled, deprotection of the final Fmoc group is carried out and the final cleavage of the peptide is carried out in the presence of a solution (10ml) of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) in DMF (1/1/8) in the presence of DIEA in acetic anhydride, after filtration, the solution is concentrated and dissolved in ether, the precipitate is centrifuged and the mother liquor is decanted, the above steps are carried out twice, the precipitate is dissolved in water and lyophilized to give the fully deprotected peptide, after lyophilization, the peptide is purified on preparative reverse-phase HP L C in the presence of 0.1% TFA by acetonitrile/water elution, the stapled peptide thus obtained preferably being more than 90% pure.

1.2 characterization:

the crosslinked compound was purified by high performance liquid chromatography on a reverse phase C18 column to give pure compound, and the chemical composition of the pure product was confirmed by L C/MS mass spectrometry and amino acid analysis.

The results are shown in table 3 below:

table 3:

example 2 evaluation of whether stapled peptides retain the α -helix structure:

in order to further understand the influence of shortened polypeptide length on α -helix structure, and utilize the characteristic that trifluoroethanol has the effect of promoting the formation of α -helix secondary structure, the stapled peptide is subjected to circular dichroism test analysis, the typical secondary structure of the polypeptide in the far ultraviolet region (185-245 nm) on a CD spectrogram has respective characteristic absorption peaks, wherein, α -helix has double negative peaks at 208nm and 222nm and shows a positive peak at 195nm, β -fold has a weaker negative peak at 215nm, and random coiling has a negative peak at 200nm and a small and wide positive peak at 220 nm.

The secondary structure of stapled peptide was determined by circular dichroism spectroscopy, CD spectra were measured using a J-810 spectrometer at 25 ℃ using a quartz cell with an optical path of 1.0mm and recorded by scanning in the range 190 to 260nm, at a speed of 50nm/min and a bandwidth of 1nm and a response of 1s, a solution of the polypeptide in deionized water (water environment) and a 50% trifluoroethanol solution prepared from PBS (membrane-simulated environment) was prepared at a concentration of 0.4mg/m L, each spectrum being the result of 3 mean accumulations, and the obtained CD spectra were then converted into mean residual ellipticity using the following formula:

[θ]＝(θobs·1000)/(c*l*n)

wherein [ theta ]]Is the residual ovality (deg.cm 2 deg.dmol)^-1) θ obs is the observed ellipticity corrected for buffer at a given wavelength (mdeg), c is the peptide concentration (mM), l is the path length (mM), and n is the number of amino acids.

α -helix content was calculated according to the following formula:

α -helix content (%) ([ theta ])]222*100)/θ_f

θ_f＝-39500*(1-2.57/n)

Wherein [ theta ]222 represents the ellipticity of an amino acid residue at 222nm, and n represents the number of amino acids contained in the polypeptide.

The resulting CD spectra show the mean residual ellipticity θ (deg cm)²*dmol^-1) For a wavelength λ (nm).

As shown in FIGS. 2B & E, Peptide-DB-2 formed a random coil structure in both aqueous environment and membrane-simulated environment, whereas in aqueous environment and 50% trifluoroethanol membrane-simulated environment, both SAH-SOS1A and Peptide-DB-1 formed a α -helix structure (FIGS. 2C & F), but as can be seen from FIGS. 2A & D, Peptide-DB-1 had a good α -helix structure in both aqueous environment and membrane-simulated environment, and the helix structure of Peptide-DB-1 in aqueous environment was significantly better than SAH-SOS1A, and the later calculated content of polypeptide α -helix in aqueous environment and membrane-simulated environment also confirms that in aqueous environment, the α -helix content of SAH-SOS1 was only 4.6%, the α -helix content of Peptide-DB-1 was only 26.1%, the helix content of Peptide-DB-1 was only 4.1%, the helix content of Peptide-DB-15% and the Peptide-DB-15-coil structure was not reduced by the comparable length of Peptide-PEG-S-DB-15-S-DB-15, respectively.

Example 3: evaluation of stapled peptides with KRAS^G12CBinding affinity of protein:

for detection of SAH-SOS1AAnd Peptide-DB-1 and KRAS^G12CDirect binding of proteins is measured in real time using the label-free biomolecule binding ForteBio Octet Red 96 system and the biolayer interferometry (B L I) technique, the biomolecules bind to the sensor surface to form a biofilm that interferes with the light transmitted through the sensor, the interference is detected by phase shift to detect changes in the number of molecules bound to the sensor surface, and the binding affinity is usually evaluated using Kd (equilibrium dissociation constant), which is used to evaluate the strength of the bimolecular interaction.

Evaluation of stapled peptides with KRAS by biofilm interference technique (B L I)^G12CThe binding affinity of the protein; KRAS was evaluated using a biolayer interferometric biosensor in an OctetRed 96 instrument^G12CBinding affinity between the protein and the peptide; all assays were performed on corning 96-well black plates, and binding data was collected at 25 ℃; first, KRAS is prepared^G12CIncubating the protein with biotin and standing at 4 ℃ for 2 hours, and removing free biotin by dialysis; in the reaction of biotin-KRAS^G12CAll biosensor tips were pre-wetted for binding by soaking in PBS buffer for 10 minutes before protein immobilization on SSA (enhanced streptavidin) biosensors (ForteBio Inc) and subtracted using a reference biosensor, the running buffer for dialysis, fixation and dilution was PBS at room temperature, and the total working volume of sample and buffer was 200 μ L, the procedure described above included five steps of (1) baseline 1, immersion of the biosensor in PBS buffer for 60s equilibration, (2) biotin-KRAS^G12CProtein loading on SSA biosensor for 600 seconds; (3) baseline 2, move biosensor to PBS buffer solution and equilibrate for 60 s; (4) binding, moving the biosensor to wells with different concentrations of peptide for 300 seconds to measure K_on(ii) a (5) Dissociation, the biosensor was immersed in PBS buffer for 300 seconds to measure K_off(ii) a Each peptide was repeated at least three times; all data were calculated using data analysis software supplied by ForteBio and according to k_onAnd k is_offThe equilibrium dissociation constant (KD) values were calculated.

Parent of SAH-SOS1A with Peptide-DB-1And force as shown in FIG. 3; Peptide-DB-1 directly with KRAS^G12CProtein binding with a kinetic dissociation constant (Kd) of 5.05 μ M, much stronger than SAH-SOS1A (Kd 46.9 μ M); compared with AH-SOS1A, Peptide-DB-1 and KRAS^G12CThe protein is more stable and binds more efficiently.

Example 4: Peptide-DB-1 reduces KRAS^G12CSurvival of mutant cells:

cell viability of H358 and A549 cells was analyzed by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay, MTT was dissolved in PBS to a stock concentration of 5mg/m L and stored at-20 deg.C, briefly, cells (7 × 10)³Individual cells/well) were seeded into 96-well plates and incubated at 37 ℃ with 5% CO₂Overnight to adhere the cells and treated with peptide at various concentrations in triplicate for 24 hours, then a 20 μ L MTT (5mg/ml) solution in PBS was added to each well in the dark and the plates were incubated at 37 ℃ for an additional 4 hours, after which the supernatant was carefully removed and formazan crystals were dissolved in 150 μ L DMSO, and after 15 minutes of shaking the absorbance was measured at 570nm with a Multiskan FC plate reader.

As can be seen from FIG. 4A, at higher concentrations (1.25. mu.M and 2.5. mu.M), Peptide-DB-1 inhibited H358 cells by 50% or more, comparable to the inhibition of the positive control SAH-SOS 1A; MTT results show that Peptide-DB-1 inhibits KRAS in a concentration-dependent manner^G12CProliferation of mutant cell H358; however, even after increasing the peptide concentration, no significant cell proliferation inhibitory activity was observed in a549 cells (fig. 4B); DB-1 has a strong inhibition on H358 cells, but has a slight inhibition on A549 cells.

Example 5: Peptide-DB-1 directly inhibits KRAS^G12CBinding to GDP:

mant-GDP and KRAS^G12CBinding of the protein was monitored by fluorescence measurements over time on a Molecular Devices iD5 multifunctional microwell plate reader (excitation 355nm, emission 448 nm); different concentrations of stapled peptide were mixed with KRAS^G12CProtein and mant-GDP were incubated at 25 ℃ in 25mM Tris (pH 7.5), 50mM NaCl and 1mM DTT buffer; KRAS^G12COf proteins and of mant-GDPThe final concentrations were all 1. mu.M; KRAS alone^G12CProtein and mant-GDP binding as a positive control, and competition with 200-fold excess unlabeled GDP as a negative control.

Peptide-DB-1 inhibits KRAS in a concentration-dependent manner in vitro^G12CBinding to GDP; reacting KRAS^G12CAnd equimolar fluorescent GDP analogue mant-GDP (2'-/3' -O- (N '-methylcarbamoyl) guanosine-5' -O-diphosphate) was used as positive control; excess unlabeled GDP (200X) was used as a negative control; then KRAS^G12CCo-incubation with mant-GDP and SAH-SOS1A or Peptide-DB-1; the decrease in relative fluorescence observed in the same curve reflects KRAS^G12CThe transforming activity of GDP in the active site; in the different curves, higher relative fluorescence values indicate a contrast to KRAS^G12CThe inhibition effect of the combination with GDP is stronger; as shown in FIG. 5, both SAH-SOS1A and Peptide-DB-1 inhibited the binding of mant-GDP in a concentration-dependent manner; Peptide-DB-1 blocking nucleotide and KRAS^G12CThe binding of (B) was also comparable to that of the positive control SAH-SOS 1A.

Example 6: Peptide-DB-1 induces H358 cell arrest at G₂In the period of/M:

KRAS mutant cells (A549 and H358) were stained with Propidium Iodide (PI) and subjected to cell cycle analysis by flow cytometry, and the cells were stained with 2 × 10⁵Cell/well, 4 × 10⁵Cell/well concentrations were seeded in 6-well plates and after 24 hours the media was replaced with media containing various concentrations of peptides (0, 10, 20 and 40 μ M) and left for another 24 hours, floating and attached cells were harvested by trypsinization, centrifuged, resuspended and fixed in 70% (v/v) ethanol overnight at 4 ℃, then cells were washed with PBS and resuspended in 0.5M L PI staining solution for 30 minutes at 37 ℃ in the dark, PI staining solution was performed, cells were filtered through 200 mesh cell sieves before determining DNA content by Flow cytometry (SQMACuant, Miltenyi Biotec GmbH), 10,000 events were collected and analyzed sparingly, cell cycle G1, S and G were calculated using Flow Jo7.6.1 software₂Percentage of cell population in the M phase.

MTT results show that Peptide-DB-1 reduces the survival rate of H358 cells, and Peptideide-DB-1 can inhibit KRAS concentration-dependently^G12CProliferation of mutant cell H358; the reduction in cell viability caused by stapled peptides may occur through two pathways: (1) over-induced apoptosis, cell necrosis, autophagy and other death pathways; (2) too-disturbed cell cycle fails cell proliferation; to investigate the mode of action of Peptide-DB-1, which resulted in a reduction in the viability of H358 cells, the effect of Peptide-DB-1 on the cell cycle distribution of H358 cells and A549 cells was examined by flow cytometry (FIG. 6A); the results showed that G was increased with increasing Peptide-DB-1 concentration after application of Peptide-DB-124H on H358 cells₂Gradual increase of cells in the/M phase; as shown in FIG. 6B, G₂the/M cell cycle arrest increased from 16.76% to 49.79%; indicating that Peptide-DB-1 induced significant G in H358 cells₂Retardation in the M phase; however, as shown in FIG. 6C, no significant G was observed when A549 cells were treated with Peptide-DB-1₂Cell cycle arrest in the/M phase; in addition, the determination of Peptide-DB-1 vs G by immunoblot analysis₂The effect of the protein level associated with the M phase to understand the mechanism of cell cycle arrest in Peptide-DB-1 induced KRAS mutant cell lines; as shown in FIG. 6D, G in Peptide-DB-1 treated H358 cells₂The key regulatory proteins cyclin B1 and CDK1 were significantly reduced during the/M phase, while no change in A549 cells was observed (FIG. 6E); the results showed that Peptide-DB-1 induced G in H358 cells₂the/M phase block was probably due to effects on cyclin B1 and CDK1 and resulted in inhibition of cell proliferation.

Example 7: Peptide-DB-1 induces H358 apoptosis:

by mixing 2 × 10⁵H358 and A549 cells were seeded in 6-well plates for apoptosis assay, after cell adhesion, cells were treated with various concentrations of peptide (0, 2.5, 10 and 20 μ M) for 24 hours, trypsinized and washed 3 times with cold PBS, then resuspended in binding buffer and cell density adjusted to 1 × 106 cells/M L, then stained with the AnnexinV-FITC apoptosis detection kit and incubated at room temperature in the dark for 20 minutes, and cell samples were assayed by flow cytometry (MACSQuant, Miltenyi Biotec GmbH)Previously, cells were filtered through a 200 mesh cell screen; at least 10,000 events were evaluated using Flow Jo _ V10 software and apoptosis was analyzed by evaluating the percentage of apoptotic cells.

To investigate whether induction of apoptosis also contributed to Peptide-DB-1 mediated growth inhibition in KRAS mutant cells, the number of apoptotic cells was analyzed using Annexin V-FITC/PI and flow cytometry; as shown in FIGS. 7A & B, the apoptosis rate of H358 cells was dose-dependent at 2.5. mu.M, 10. mu.M and 20. mu.M Peptide-DB-1 concentrations, 56.63%, 61.40% and 70.70%, respectively, and significantly higher than that of the negative control (36.42%); the apoptosis rate of a549 cells was 28.80%, 29.13% and 36.97%, respectively (fig. 7A & C), and the overall incidence was not significantly higher than that of the negative control group (26.63%); to further demonstrate the mechanism by which Peptide-DB-1 induces apoptosis in KRAS mutant cell lines, western blot analysis was performed to assess the expression of several well characterized apoptotic proteins; as shown in FIGS. 7D & E, Peptide-DB-1 has no obvious effect on the expression of the pro-apoptotic protein Bax, but effectively inhibits the expression of the anti-apoptotic protein Bcl-2 in H358 cells and increases the expression of Caspase 3; in A549 cells, no significant increase or decrease in apoptotic proteins was observed, further confirming that induction of apoptosis is also one of the causes of Peptide-DB-1 in inhibiting H358 cell proliferation.

Example 8: Peptide-DB-1 enhances enzymatic stability:

NH of 3. mu.g/m L trypsin and α -chymotrypsin at 37 ℃₄HCO₃150 μ g/m L of Peptide-DB-1 was enzymatically degraded in buffer (Peptide to enzyme mass ratio 50:1), 50 μ L samples were taken after 0 min, 30 min, 1 h, 2h, 6 h, 12h and 24h, the enzymatic reaction was stopped by adding 1% (v/v) formic acid in equal amounts, the samples were then analyzed by Shimadzu HP L C, the chromatographic separation was performed using a Thermo scientific Syncronism TMC18 column (250 × 4.6.6 mm, 5 μm) with water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B), the gradient program was 0 min 20% B, 30 min 90% B, 31 min 20% B, 40 min 20% B, the flow rate was 1m L min^-1All samples were taken at 20. mu. L.

Since most peptides have short half-lives, the enzymatic stability of Peptide-DB-1 was first investigated, NH at 37 ℃ in the presence of trypsin or α -chymotrypsin₄HCO₃The stability of Peptide-DB-1 enzyme was investigated in buffer, and the HP L C chromatogram of enzymatic degradation of Peptide-DB-1 is shown in FIG. 8A&B is shown in the specification.

Trypsin cleaves peptide chains predominantly on the carboxy side of lysine or arginine; since only tyrosine is present in the amino acid sequence of Peptide-DB-1, trypsin attacks only tyrosine, and it is presumed that FGIS is gradually produced₅LTNS₅L K fragment, the peak value of Peptide-DB-1 continued to decrease with the progress of degradation, after 24 hours of degradation, 6% of Peptide-DB-1 remained undegraded, the degradation product of trypsin reached 78% in 12 hours, and after 24 hours of degradation, the degradation product of trypsin was only 34%, indicating that the degradation product of trypsin was degraded again (FIG. 8C)&E)。

α -chymotrypsin preferentially cleaves Peptide amide bonds with tyrosine, tryptophan or phenylalanine on the carboxy side of the amide bond, the amino acid sequence of Peptide-DB-1 contains only phenylalanine, so α -chymotrypsin attacks mainly phenylalanine, the peak value of Peptide-DB-1 continues to decrease as degradation proceeds, but no distinct peak characteristic of α -chymotrypsin degradation products is observed, 70% of Peptide-DB-1 remains without degradation after 24 hours of degradation, and Peptide-DB-1 has better stability in α -chymotrypsin (FIG. 8D & F).

Example 9: Peptide-DB-1 enhances its stability in plasma:

mixing 500 μ L rat plasma with the Peptide-DB-1 solution to a final concentration of 500 μ g/m L, incubating the mixture at 37 deg.C, taking aliquots of 50 μ L solution at different time intervals and mixing with 150 μ L methanol-acetonitrile (50:50, v/v) to precipitate plasma proteins, vortexing and centrifuging at 12,000rpm for 20 minutes at 4 deg.C, transferring the supernatant and assaying with Shimadzu HP L C, and chromatographing using Thermo Scientific^TMSyncronis^TMC18 chromatography column (250 × 4.6.6 mm, 5 μm) with mobile phase containing 0.1% trifluoro benzeneWater of acetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B) by a gradient procedure of 0 min 20% B, 30 min 90% B, 31 min 20% B, 40 min 20% B, flow rate 1m L min^-1All samples were taken at 20. mu. L.

The stability of Peptide-DB-1 was found to be good by enzyme stability experiments, and therefore, the stability of Peptide-DB-1 and SAH-SOS1A was tested in plasma stability experiments, the stability of SAH-SOS1A and Peptide-DB-1 was measured in rat plasma after time-dependent incubation with HP L C, as shown in FIG. 9A, the peak time of SAH-SOS1A and Peptide-DB-1 was 16.0999min and 21.241 min, respectively, in FIG. 9C, the characteristic peak of SAH-SOS1A was not observed in the 0 minute chromatogram, and it is presumed that SAH-SOS1A degraded after plasma addition, whereas Peptide-DB-1 was stable in rat plasma (FIGS. 9D & F), and at the same time, the peak area of Peptide-DB-1 began to increase after 8 hours of incubation in plasma.

Claims (10)

1. A stapled peptide comprising the amino acid sequence A₀B₀C₀D₀E₀F₀G₀A₁B₁C₁D₁Wherein: a. the₀Is F or a conservative substitution; b is₀Is G or a conservative substitution; c₀Is I or a conservative substitution; d₀Is Y or a conservative substitution; e₀L or a conservative substitution F₀Is T or a conservative substitution; g₀Is N or a conservative substitution; a. the₁Is I or a conservative substitution; b is₁L or a conservative substitution C₁Is K or a conservative substitution; d₁Is T or a conservative substitution;

the stapled peptide comprising an α -helix and two unnatural amino acids X cross-linked at the i and i +3 positions, or at the i and i +4 positions, or at the i and i +7 positions by a macrocycle-forming linker;

the stapled peptide is selected from Ac-FGIX L TNX L KT-NH₂、Ac-FFGIXLTNXLKTE-NH₂(ii) a Wherein two unnatural amino acids X are end-capped with an olefin and cross-linked through a macrocycle-forming linker.

2. The stapled peptide of claim 1 wherein said two unnatural amino acid X is (S) - α -methyl- α -pentenylglycine.

3. The stapled peptide of claim 1 wherein said amino acid sequence is Ac-FGIX L TNX L KT-NH₂。

4. The stapled peptide as claimed in any of the preceding claims 1 to 3, wherein the two unnatural amino acids X are cross-linked at the i and i +4 positions by a macrocycle-forming linker.

5. The stapled peptide of claim 1 wherein the amino acid sequence is 11 amino acids in length; and the position of the unnatural amino acid is fixed.

6. A pharmaceutical composition comprising a stapled peptide of claim 1.

7. A pharmaceutical composition according to claim 6, wherein said pharmaceutical composition comprises a drug for the treatment of cancer.

8. A method of treating cancer, comprising administering to a patient in need thereof a stapled peptide of claim 1.

9. The method of claim 8, wherein the cancer is non-small cell lung cancer; said is notPatients with small cell lung cancer harboring mutant KRAS^G12CA protein.

10. The stapled peptide of claim 1 wherein said stapled peptide selectively inhibits H358 cells;

the stapled peptides have anti-proteolytic ability and plasma stability.

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