DESC:TECHNICAL FIELD
The present disclosure is in the field of peptide mimics. In particular, the disclosure relates to short peptides that mimic alpha-helical structure of Helix 4 of the DNA-binding domain of ERG transcription factor.

BACKGROUND OF THE DISCLOSURE
Prostate cancer (PCa) is the second most common projected cancer incidence (14.1% of 10.1 million cases) among males worldwide and third (6.1% of 679,421 cases) in India. PCa is a highly heterogeneous disease associated with several types of genomic alterations and has been categorized into different molecular subclasses comprising ETS-fusion positive (overexpression of ERG, ETV1, ETV4, or other ETS (E26 transformation-specific) family transcription factors), SPOP/CHD1 altered and ETS-fusion negative subtype. More than 50% of PCa cases harbour the most recurrent TMPRSS2-ERG gene fusion, i.e., between the 5'-untranslated region of androgen-regulated TMPRSS2 (transmembrane protease, serine 2) and coding region of ERG (v-ets erythroblastosis virus E26 oncogene homolog). TMPRSS2-ERG gene fusion is a common genetic alteration leading to ERG oncoprotein overexpression, which plays an important role in early-pathogenesis of this disease. Patients with TMPRSS2-ERG fusion show a much higher rate of recurrence than those without fusion.

It has been reported that conditional ERG transgenic mice with Pten heterozygous loss background developed invasive prostatic adenocarcinoma rapidly. Furthermore, knockdown of TMPRSS2-ERG fusion product inhibited cell proliferation, migration and tumor growth in mice. Overexpression of ERG in PCa has been known to induce global changes in chromatin conformation. Genome-wide binding analysis revealed the existence of a highly integrated transcriptional network of ERG and androgen receptor (AR). Moreover, ERG disrupts AR signaling by inducing histone repressive marks via direct activation of the Polycomb Group protein, EZH2, which catalyzes H3K27 trimethylation. Another study has suggested that AR and ERG transcriptional complex, together with epigenetic regulators HDACs and EZH2, functions to mediate repression of cytoskeletal genes that promote epithelial differentiation and inhibit metastasis. This molecular evidence suggests that ERG in combination with oncogenic corepressors including HDACs and the polycomb protein, EZH2, could impede epithelial differentiation, thus contributing to PCa progression. Recently, it has been shown that ~96% of TMPRSS2-ERG fusion-positive and ~70% of AR-positive patients show elevated DLX1 expression and ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. Inhibitory peptides targeting DNA binding domain of ERG are known to impair its chromatin recruitment, thereby resulting in reduced proliferation and invasion of TMPRSS2-ERG fusion positive PCa cells. Collectively, targeting ERG oncoprotein has high therapeutic relevance as till date no therapeutic modality has been approved for clinical management of ERG-positive PCa patients. Therefore, providing small peptides that inhibit binding of ERG transcription factor with its target DNA sequences are expected to aid in the early-stage management of prostate cancer.

Furthermore, the main therapy for clinical management of advanced PCa often includes ablation of androgen biosynthesis or AR activity using pharmacological inhibitors; however, tumors invariably develop adaptive mechanisms, many of which restore androgen signaling and expression of ERG oncoprotein in tumors that harbour TMPRSS2-ERG fusion. Although TMPRSS2-ERG positive PCa represents the major molecular subtype, targeting ERG transcription factor remains challenging. Therefore, therapeutic targeting of ERG transcription factor is of prime clinical significance for the treatment and management of PCa patients. The present disclosure attempts to address this need.

SUMMARY OF THE DISCLOSURE
The present disclosure relates to a peptide having Formula (I):

(I)
wherein,
P is a protecting group;
-CH2-CH2-CH2- is a propyl linker that is connected to the backbone nitrogen atom of X at one end and to the backbone nitrogen atom of the a-amino group of glycine (G) at the other end;
X is an amino acid, the backbone amino group nitrogen (N) of which is covalently connected to a CH2 group of the propyl linker and to the protecting group as shown in the above formula;
X2 is an amino acid selected from glutamic acid and aspartic acid;
X3 is an amino acid selected from lysine, arginine, ornithine, and histidine;
G is glycine, the backbone nitrogen of which is covalently connected to a CH2 group of the propyl linker;
X4 is an amino acid selected from serine, threonine, aspartic acid, glutamic acid, lysine, histidine, alanine, and leucine;
X5 is an amino acid selected from arginine, lysine, ornithine, and histidine;
X6 is an amino acid selected from alanine, valine, leucine and isoleucine;
X7 is an amino acid selected from glutamic acid, aspartic acid, lysine, serine, alanine, leucine and isoleucine;
X8 is an amino acid selected from arginine and lysine;
X9 is an amino acid selected from tyrosine and phenylalanine;
X10 is an amino acid selected from tyrosine and phenylalanine;
X11 is an amino acid selected from tyrosine and phenylalanine;
Z is an amino acid;
n = 0, 1, 2, or 3;
R is -OR1 or -NR2R3,
R1 is H or alkyl;
each of R2 and R3 is independently H or alkyl;
and
wherein the peptide mimics Helix 4 of the DNA-binding domain of ERG transcription factor.

The present disclosure also provides a conjugate comprising the peptide of Formula (I), wherein the peptide is linked to a moiety at the N or C terminus of the peptide.

The present disclosure further provides a pharmaceutical composition comprising the peptide of Formula (I) or the conjugate containing the peptide of Formula (I) and a pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Figure 1, panels A and B show different regions of the Total Correlated Spectroscopy (TOCSY) spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe (1).
Figure 2 shows a Heteronuclear Single Quantum Coherence (HSQC) spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe (1).

Figure 3 shows the 1H-NMR spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe (1).

Figure 4 shows shows the 13C-NMR spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-Ome.

Figure 5, panel a) shows the temperature-dependent (278-353 K) CD spectra of Moc-[GE(O-tBu)K(Boc)]G-OMe in 10 mM Tris-chloride aqueous buffer (pH = 7.4); and panel b) shows the temperature-dependent (278-353 K) CD spectra of Moc-[GE(O-tBu)K(Boc)]G-OMe in 10% TFE in 10 mM Tris-chloride aqueous buffer (pH = 7.4).

Figure 6, panel A shows the MALDI spectra of helicomimic ERG-A (Moc-[GEK]G-SRA-LRYYY-DK-NH2); and panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Figure 7, panel A shows the MALDI spectra of helicomimic ERG-B (Moc-[GEK]G-ERA-LRYYY-DK-NH2); and panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Figure 8 shows the MALDI spectra of helicomimic ERG-C (Moc-[GEK]G-SRA-ERYYY-DK-NH2).

Figure 9, panel A shows the MALDI spectra of the acyclic analogue ERG-D (Ac-YDKLSRALRYYYDKN-NH2) and panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Figure 10: Panel a) shows the temperature-dependent (278-358 K) CD spectra of ERG-A (Moc-[GEK]G-SRA-LRYYY-DK-NH2) in 10 mM Tris-chloride aqueous buffer (pH = 7.4), Panel b) shows the temperature-dependent (278-353 K) CD spectra of ERG-B (Moc-[GEK]G-ERA-LRYYY-DK-NH2) in 10 mM Tris-chloride aqueous buffer (pH = 7.4), Panel c) shows the temperature-dependent (278-353 K) CD spectra of ERG-C (Moc-[GEK]G-SRA-ERYYY-DK-NH2) in 10 mM Tris-chloride aqueous buffer (pH = 7.4).

Figure 11: UV-Vis Absorbance vs wavelength plots of ERG-ds-DNA titration with: Panel a) ERG-A, Panel b) ERG-B, Panel c) ERG-C, Panel d) ERG-D, in 10 mM Tris-chloride aqueous buffer (pH = 7.4). Plots of A0/A- A0 (where A0 and A are the absorbances of the DNA and its complex with peptide, respectively) vs 1/[peptide] plots of ERG-A, ERG-B, ERG-C and ERG-D are shown on the right side of each spectrum.

Figure 12: Fluorescence Intensity vs wavelength plots of ERG-FAM-ds-DNA titration with: Panel a) ERG-A, Panel b) ERG-B, Panel c) ERG-C, Panel d) ERG-D, in 10 mM Tris-chloride aqueous buffer (pH = 7.4). Plots of 1/F0-F (where Fo-F is the difference in the fluorescence intensity of FAM tagged ds-DNA in native state and in complex with peptide) vs 1/[peptide] plots of ERG-A, ERG-B, ERG-C and ERG-D are shown on the right side of each spectrum.

Figure 13 shows plots of Microscale Thermophoresis (MST) showing fraction of Fluorescently-tagged ds-DNA bond with peptide (ERG-A, ERG-B, ERG-C, ERG-D) vs peptide concentration.

Figure 14 shows plots of Isothermal Titration Calorimetry (ITC) showing kcal mol-1 of injectant vs [peptide/DNA] of ERG-A, ERG-B, ERG-C, ERG-D.

Figure 15 shows the LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-H-FNT-001.

Figure 16 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-H-FNT-002.

Figure 17 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-P-FNT-001.

Figure 18 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-RP-FNT-001.

Figure 19 shows the MALDI spectra for ERG-NC-FNT-001 (A) and the corresponding HPLC chromatogram (B).

Figure 20 shows the MALDI spectra for ERG-NC-FNT-002 (A) and the corresponding HPLC chromatogram (B).

Figure 21 shows the MALDI spectra for ERG-NC-FNT-003 (A) and the corresponding HPLC chromatogram (B).

DETAILED DESCRIPTION OF THE DISCLOSURE
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having”, or “including but not limited to” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Reference throughout this specification to “one embodiment”, “an embodiment”, “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” “in an embodiment”, on “in some embodiments” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

As used herein, the term “helicomimic” refers to a peptide that comprises a propyl linker acting as a hydrogen bond surrogate (HBS) between the first and the fourth amino acid of the peptide, wherein the HBS provides a stable alpha-helical conformation to the peptide. The alpha-helical conformation of the HBS-containing peptide mimics the natural alpha-helical conformation of Helix 4 of the DNA-binding domain of the ERG transcription factor. The terms “helicomimic”, “peptide helicomimic”, “HBS helicomimic”, “peptide of Formula (I)” and “peptide mimic” are used interchangeably throughout the disclosure.

The term “about” as used herein encompasses variations of +/- 10% and more preferably +/- 5%, as such variations are appropriate for practicing the present invention.

The present inventors have developed peptide helicomimics that mimic Helix 4 of the DNA-binding domain of ERG transcription factor and selectively bind to double-stranded (ds) DNA sequences recognized by ERG transcription factor.

The DNA-binding domain of human ERG contains five helices, out of which three are well defined and two are short and partial helices. Helix 4 (H4) makes the maximum number of interactions with DNA. The DNA-binding domain of H4 contains 14 amino acid residues. Using X-ray crystallographic analysis, the inventor determined that 11 out of 14 amino acid residues in Helix 4 interact with the nucleic acid bases in the major groove of dsDNA.

The amino acid sequence of Helix-4 of the DNA-binding domain of human ERG is:
356Ser-Lys-Pro-Asn-Met-Asn-Tyr-Asp-Lys-Leu-Ser-Arg-Ala-Leu-Arg-Tyr-Tyr-Tyr-Asp-Lys-Asn376 (SEQ ID NO: 1).

The amino acid sequence of the dsDNA binding segment of Helix-4 is:
Asp363-Lys364-Leu365-Ser366-Arg367-Ala368-Leu369-Arg370-Tyr371-Tyr372-Tyr373-Asp374-Lys375-Asn376 (SEQ ID NO: 2).

Out of 11 amino acid residues that interact with the dsDNA, the inventor determined that the amino acid residues shown in bold in SEQ ID NO: 2 make important interactions with the dsDNA. Based on this analysis, the inventor designed hydrogen bond surrogate (HBS) constrained peptide helicomimics of Helix 4 of the DNA-binding domain of ERG.

The peptide helicomimics of the present disclosure contain about 12-15 amino acids, wherein the first and the fourth amino acids are connected by a propyl linker that forces an a-helical fold into the peptide.

The present disclosure provides a peptide having Formula (I):

(I)
wherein,
P is a protecting group;
-CH2-CH2-CH2- is a propyl linker that is connected to the backbone nitrogen atom of X at one end and to the backbone nitrogen atom of the a-amino group of glycine (G) at the other end;
X is an amino acid, the backbone amino group nitrogen (N) of which is covalently connected to a CH2 group of the propyl linker and to the protecting group as shown in the above formula;
X2 is an amino acid selected from glutamic acid and aspartic acid;
X3 is an amino acid selected from lysine, arginine, ornithine, and histidine;
G is glycine, the backbone nitrogen of which is connected to a CH2 group of the propyl linker;
X4 is an amino acid selected from serine, threonine; aspartic acid, glutamic acid, lysine, histidine, alanine, and leucine;
X5 is an amino acid selected from arginine, lysine, ornithine, and histidine;
X6 is an amino acid selected from alanine, valine, leucine and isoleucine;
X7 is an amino acid selected from glutamic acid, aspartic acid, lysine, serine, alanine, leucine and isoleucine;
X8 is an amino acid selected from arginine and lysine;
X9 is an amino acid selected from tyrosine and phenylalanine;
X10 is an amino acid selected from tyrosine and phenylalanine;
X11 is an amino acid selected from tyrosine and phenylalanine;
Z is an amino acid;
n = 0, 1, 2, or 3;
R is -OR1 or -NR2R3,
R1 is H or alkyl;
each of R2 and R3 is independently H or alkyl;
and
wherein the peptide mimics Helix 4 of the DNA-binding domain of ERG transcription factor.

The peptide of Formula (I) is a helicomimic and contains about 12-15 amino acids. The propyl linker between the backbone nitrogen atom of the first amino acid (X) at one end and the backbone nitrogen atom of the a-amino group of glycine (G) at the other end acts as a hydrogen bond surrogate and forces the peptide predominantly into a stable a-helix.

The backbone nitrogen atom of the first amino acid (X) is connected to P which is a protecting group. In some embodiments, the protecting group is selected from methoxycarbonyl (Moc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), Allyloxycarbonyl (Alloc), fluorenylmethoxycarbonyl (Fmoc), and nosyl (Ns). In some embodiments, the protecting group is Moc. It would be apparent to one of ordinary skill in the art that the protecting group need not be restricted to those listed here and any other protecting group can also be employed in the peptides of Formula (I).

In the peptide of Formula (I), -CH2-CH2-CH2- is a propyl linker that is connected to the backbone nitrogen atom of X at one end and to the backbone nitrogen atom of the a-amino group of glycine (G) at the other end. The -CH2-CH2-CH2- linker, also referred to herein as a propyl linker, acts as a hydrogen bond surrogate (HBS) and facilitates the alpha-helical configuration of the peptide.

In the peptide of Formula (I), X is an amino acid, the backbone amino group nitrogen (N) of which is covalently connected to a CH2 group of the propyl linker and to P as shown in the above formula. In some embodiments, X can be any amino acid. In some embodiments, X is glycine or alanine. In some embodiments, X is glycine.
The carbonyl group of X is covalently connected to the amino group of the next amino acid, X2; the carbonyl group of X2 is covalently connected to the amino group of the next amino acid, X3; the carbonyl group of X3 is covalently connected to the alpha-amino group of glycine (G); the carbonyl group of G is covalently connected to the amino group of the next amino acid, X4 and so on. Thus, in the peptide of Formula (I), the alpha-amino group of G is covalently connected to a CH2 group of the propyl linker and to the carbonyl group of X3.

X2 is an amino acid selected from glutamic acid (Glu) and aspartic acid (Asp). In some embodiments, X2 is glutamic acid.

X3 is an amino acid selected from lysine (Lys), arginine (Arg), ornithine (Orn), and histidine (His). In some embodiments, X3 is lysine.

As noted above, in Formula (I), G is glycine, the backbone nitrogen of which is connected to a CH2 group of the propyl linker and to the carbonyl group of X3.

X4 is an amino acid selected from serine (Ser), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), histidine (His), alanine (Ala), and leucine (Leu). In some embodiments, X4 is serine or glutamic acid. In some embodiments, X4 is serine.

X5 is an amino acid selected from arginine (Arg), lysine (Lys), ornithine (Orn), and histidine (His). In some embodiments, X5 is arginine.

X6 is an amino acid selected from alanine (Ala), valine (Val), leucine (Leu), and isoleucine (Ile). In some embodiments, X6 is alanine.

X7 is an amino acid selected from glutamic acid (Glu), aspartic acid (Asp), lysine (Lys), serine (Ser), alanine (Ala), leucine (Leu) and isoleucine (Ile). In some embodiments, X7 is leucine or glutamic acid. In some embodiments, X7 is glutamic acid.

X8 is an amino acid selected from arginine (Arg) and lysine (Lys). In some embodiments, X8 is arginine.

X9 is an amino acid selected from tyrosine (Tyr) and phenylalanine (Phe). In some embodiments, X9 is tyrosine.

X10 is an amino acid selected from tyrosine (Tyr) and phenylalanine (Phe). In some embodiments, X10 is tyrosine.

X11 is an amino acid selected from tyrosine (Tyr) and phenylalanine (Phe). In some embodiments, X11 is tyrosine.

In some embodiments of Formula (I), X is glycine, X2 is glutamic acid, X3 is lysine, X4 is serine or glutamic acid, X5 is arginine, X6 is alanine, X7 is leucine or glutamic acid, X8 is arginine, X9 is tyrosine, X10 is tyrosine, and X11 is tyrosine.

In some embodiments of Formula (I), n is zero and Z is absent.

In some embodiments of Formula (I), n is 1, and Z is represented as X12, wherein X12 is an amino acid selected from aspartic acid (Asp) and glutamic acid (Glu). In some embodiments, X12 is aspartic acid.

In some embodiments of Formula (I), n is 2, and Z is represented as X12-X13, wherein X12 is an amino acid selected from aspartic acid and glutamic acid and X13 is an amino acid selected from lysine and arginine. In some embodiments, when n = 2, Z is X12-X13, wherein X12 is aspartic acid and X13 is lysine. In the embodiments where n is 2 and Z is represented as X12-X13, the peptide of Formula (I) can be represented by Formula (II):

(II).

In some embodiments of Formula (I), n is 3, and Z is represented as X12-X13-X14, wherein X12 is an amino acid selected from aspartic acid and glutamic acid, X13 is an amino acid selected from lysine and arginine, and X14 is any amino acid. In some embodiments, X14 is asparagine (Asn). In some embodiments, when n = 3, Z is X12-X13-X14, wherein X12 is aspartic acid, X13 is lysine, and X14 is any amino acid or asparagine. In the embodiments where n is 3 and Z is represented as X12-X13-X14, the peptide of Formula (I) can be represented by Formula (III):

(III)
In Formula (I), (II), or (III), R is -OR1 or -NR2R3.

In -OR1, R1 is H or alkyl. When R1 is alkyl, -OR1 is an alkoxy group. In some embodiments of Formula (I), (II), or (III), R is -OMe; i.e., methoxy.

In -NR2R3, each of R2 and R3 is independently H or alkyl.

In some embodiments of Formula (I), P is Moc and R is -OR1. In these embodiments, the peptide of Formula (I) can be represented by Formula (IV):
(IV).

In some embodiments, the peptide of Formula (I) is selected from Table A below:

The peptide of Formula (I) of the present disclosure mimics Helix 4 of ERG transcription factor and binds to a double stranded (ds) DNA sequence recognized and bound by ERG. In some embodiment, the peptide of Formula (I) shows a binding affinity (Kd) of about 50-450 nM to the dsDNA sequence recognized and bound by ERG transcription factor.

When the peptide of Formula (I) binds to the dsDNA sequence recognized and bound by ERG transcription factor, the recognition of the dsDNA sequence by the peptide and its binding to the dsDNA sequence is not confined to one strand alone; instead, the recognition and binding take place between the peptide and both strands of the dsDNA. That is, the nucleotide bases of the dsDNA that interact with the peptide of Formula (I) are from both the strands of the dsDNA, not only from the same strand.

In some embodiments, the dsDNA sequence recognized and bound by the peptide of Formula (I) comprises a sequence of 5’-GAGGA-3’ and 3’-CTAC-5’, wherein in the 3’-CTAC-5’ sequence, CT is complementary to the last two nucleotides GA in the 5’-GAGGA-3’ sequence.

In some embodiments, the peptide of Formula (I) binds to the dsDNA sequence recognized and bound by ERG transcription factor with a binding affinity represented in the form of dissociation constant (Kd) of about 50-450 nM, including values and ranges thereof, such as about 50-400 nM, about 50-350 nM, about 50-300 nM, about 50-250 nM, about 50-200 nM, about 50-150 nM, about 50-100 nM, about 100-450 nM, about 100-400 nM, about 100-350 nM, about 100-300 nM, about 100-250 nM, about 100-200 nM, about 150-450 nM, about 150-400 nM, about 150-350 nM, about 150-300 nM, about 150-250 nM, about 200-450 nM, about 200-400 nM, about 200-350 nM, about 200-300 nM, about 250-450 nM, about 250-400 nM, about 250-350 nM, about 300-450 nM, about 300-400 nM, or about 350-450 nM. In some embodiments, the peptide of Formula (I) binds to the dsDNA sequence recognized and bound by ERG transcription factor with a binding affinity (Kd) of about 70-85 nM.

In some embodiments, the peptide of Formula (I) comprises a tracer dye covalently attached to one of the amino acids of the peptide. In some embodiments, the tracer dye is covalently attached to amino acid X5, X6, X7, X8, X9, X10, X11, X12, X13, or X14. In some embodiments, the tracer dye is covalently attached to amino acid X12, X13, or X14. In some embodiments, the tracer dye is a fluorescent dye, including, but not limited to, rhodamine B.

The present disclosure also provides a conjugate comprising the peptide of Formula (I), wherein the peptide is linked to a moiety at the N or C terminus of the peptide. In some embodiments, the moiety is selected from an activator, a repressor, or a nuclear localization sequence (NLS).

Depending on the moiety attached to the peptide of Formula (I), the conjugate will exhibit a desired effect. For example, in some embodiments, when an activator moiety is attached to the peptide of Formula (I), the binding of the peptide of Formula (I) to its target ds-DNA sequence (e.g., the dsDNA sequence recognized and bound by ERG) will activate the transcription of a desired gene and when a repressor moiety is attached to the peptide of Formula (I), the binding of the peptide of Formula (I) to its target ds-DNA sequence will repress the transcription of a desired gene.

In some embodiments, the activator moiety is selected from Isopropyl ß-D-1-thiogalactopyranoside (IPTG), MarA transcriptional activator (Journal of Molecular Biology, 2000, 299, 1245-1255), XylS protein (JMB, 2008, 375, 59-69), MYB activators (Front Plat Sci. 2020, 11, 941), MalT Domain III activator (Structure, 2001, 9, 1051-1060), CAP or cAMP receptor protein activator (JMB, 1999, 293, 2, 199-213) and the like.

In some embodiments, the repressor moiety is selected from Met Repressor, trp repressor (Phil. Trans. R. Soc. Lond. B 1996, 351, 527-535, Phillips and Stockley), Pti4 repressor (Mini Review, Patrick Boyle, Charles Depres. Plant Signaling & Behaviour, 5:6, 629-634, 2010), lac operon repressor (references from the Book, Joan Slonczewski and John Watkins, Microbilogy: An Evolving Science, New York, W W Norton, 2009 print); the L-arabinose operon repressor (Voet Donald, Voet Judith, Hoboken NJ, Biochemistry - Text Book, 2011, John Wiley & sons), and the like.

In some embodiments, the NLS linked to the N- or C-terminus of the peptide comprises a sequence such as PKKKRKV (SEQ ID NO: 16) or a HIV-TAT sequence, e.g., YGRKKRRQRRR (SEQ ID NO: 17). The NLS sequence attached to the peptide helps in internalization of the peptide into host cell nuclei where the peptide can selectively bind to its target dsDNA sequence.

The present disclosure also provides a pharmaceutical composition comprising the peptide of Formula (I) or the conjugate described above and a pharmaceutically acceptable excipient.

The peptide of Formula (I), the conjugate, and the pharmaceutical composition of the present disclosure can be used as therapeutic agents in treatment of diseases associated with overexpression or under expression of ERG transcription factor. For example, in some embodiments, the peptide of Formula (I), the conjugate, and the pharmaceutical composition of the present disclosure can be used as therapeutic agents in treatment of ERG-positive prostate cancer.

The peptide of Formula (I), the conjugate, and the pharmaceutical composition of the present disclosure can be used as therapeutic agents in diseases where an activation or inhibition of a target gene having a binding site for ERG transcription factor is desired. In some embodiments, the peptide mimics of the present disclosure or the conjugates thereof can be used as novel therapeutic alternatives to siRNA for selectively binding to gene promoter sequences recognized and bound by ERG to suppress the expression of genes that undergo uncontrolled expression, which results in diseases like cancer. The present peptide mimics are expected to perform better than siRNA for suppressing gene overexpression since: a) the peptide mimics do not have the problem of unintended reverse transcription into host DNA sequence; b) the peptide mimics do not add unnatural RNA particles in the nucleus or the cytosol, which can cause cell function aberrations; and c) when conjugated to an appropriate NLS sequence, the mimics can have much more selective and efficient cell internalization than siRNAs.
It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES

Example 1: Design of the peptide mimics
The amino acid sequence of Helix-4 of the DNA-binding domain of human ERG is:
356Ser-Lys-Pro-Asn-Met-Asn-Tyr-Asp-Lys-Leu-Ser-Arg-Ala-Leu-Arg-Tyr-Tyr-Tyr-Asp-Lys-Asn376 (SEQ ID NO: 1).

The amino acid sequence of the dsDNA binding segment of Helix-4 is:
363Asp-Lys-Leu-Ser-Arg-Ala-Leu-Arg-Tyr-Tyr-Tyr-Asp-Lys-Asn376 (SEQ ID NO: 2).

The inventor determined that the amino acid residues shown in bold in SEQ ID NO: 2 make important interactions with the dsDNA. Based on this analysis, the inventor designed HBS peptide helicomimics of Helix 4 of the DNA-binding domain of ERG by adding glycine before Asp363 and by replacing Leu365 with glycine and placing the hydrogen bond surrogate between these two glycine residues. Additional mimics were generated by mutating other amino acid residues in SEQ ID NO: 2. Table 1 summarizes the prepared mimics.
Table 1: Peptide mimics for ds-DNA binding
Native Alpha helical sequence HBS-a-helicomimetics
363DKLSRALRYYYDK375

(SEQ ID NO: 2).
1. Moc-[GEK]G-SRA-LRYYY-DK (designated as ERG-A in the following experiments) (SEQ ID NO: 6).
2. Moc-[GEK]G-ERA-LRYYY-DK (ERG-B) (SEQ ID NO: 5).
3. Moc-[GEK]G-SRA-ERYYY-DK (ERG-C) (SEQ ID NO: 4).

A peptide based on the native sequence – 363DKLSRALRYYYDK375 (SEQ ID NO: 2) – was also synthesized and is designated as ERG-D. ERG-D has a structure of Acetyl-YDKLSRALRYYYDKN-NH2 (SEQ ID NO: 15) and does not include a propyl linker as a hydrogen bond surrogate.

Example 2: Synthesis of the designed mimics
For the description purposes, the synthesis of the mimics is divided into two parts: (1) the synthesis of “Moc-[GE(O-tBu)K(Boc)]G-OMe (1)” which contains the first 4 residues of the helicomimic (the first 4 residues provide the alpha-helicity) and (2) the synthesis of the extended helicomimic.
Part 1: Synthesis of HBS-constrained cyclic N-carbamyl single a-helical turn analogue (HBS-constrained single helical turn peptide mimic containing the first 4 residues):

Scheme 1

Synthesis of methyl 2-(2-nitrophenylsulfonamido)acetate (Ns-Gly-OMe) :

To a cold (0 °C) stirring mixture of methylglycinate hydrochloride (1.00 gm, 7.96 mmol) and nosyl chloride (1.94 gm, 8.76 mmol) in DCM (24 ml) was added trimethylamine (TEA) (2.20 ml, 15.92 mmol) drop-wise and the mixture was stirred at room temperature until TLC indicated the complete consumption of the starting material. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (15 mL), washed with water (2 x 5 mL) and 1N HCl solution (2 X 5 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography (EtOAc:Hexane -2:3) to yield the desired product (Ns-Gly-OMe, 2a) as a white solid (1.85 gm, 6.754 mmol, 85% yield); m.p. 110 °C; (TLC-EtOAc –Rf= 0.8);¹H NMR (400 MHz, CDCl3, 10 mM) d ppm: 8.11-8.08 (m, 1H, HAroNs), 7.95-7.93 (m, 1H, HAroNs), 7.76-7.73 (m, 2H, HAroNs), 6.03 (s, 1H, HNGly), 4.02 (d, J = 5.86 Hz, 2H, HaGly), 3.61 (s, 3H, HMeOMe); HRMS m/z Calculated for C9H10N2O6SNa 297.0157, Found 297.0161.

Synthesis of methyl 2-((tert-butyloxycarbonyl)(3- hydroxypropyl)amino)acetate:

To a cold (0°C) stirring mixture of Ns-Gly-OMe (2a) (1.4 gm, 5.1 mmol) and triphenylphosphine (PPh3) (2.04 gm, 7.6 mmol) in dry THF (10 ml) under N2 atmosphere was added 1,3-propane diol (740 µl, 10.2 mmol) drop-wise followed by diisopropyl azodicarboxylate (DIAD) (1.51 ml, 7.6 mmol). After 15 mins of stirring the ice bath was removed and the reaction was continued at ambient conditions until TLC indicated the complete consumption of the starting material. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (10 mL) and washed with water (2 X 5 mL). The organic layer was concentrated to get a residue which was immediately subjected to denosylation reaction conditions by the addition of thiophenol (470 µl, 6.87 mmol) and potassium carbonate (K2CO3) (576 mg, 4.18 mmol) in acetonitrile (3 ml). The reaction was continued at ambient conditions until TLC indicated the complete consumption of the starting material. After that the solvent was removed and the resulting residue was acidified using 1N HCl (10 ml). The aqueous mixture was thoroughly washed with diethyl ether (3 x 5 ml) to remove all the water-insoluble organic impurities.

The resulting aqueous solution was then treated with K2CO3 (576 mg, 4.18 mmol) followed by di-tert-butyldicarbonate (1.05 ml, 4.58 mmol) and the reaction mixture was stirred at ambient conditions until TLC indicated the complete consumption of the starting material. Removal of the solvent resulted in a residue which was dissolved in EtOAc (15 mL), washed with water (2 x 5 ml) and the organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography (EtOAc:Hexane -1:10) to yield the desired product as a viscous oil (826 mg, 2.92 mmol, 70% yield); (TLC-EtOAc:Hexane (1:0) –Rf= 0.5);¹H NMR (400 MHz, CDCl3, 10 mM) d ppm: (mixture of two rotamers) 3.86 (s) & 3.96 (s) (2H, HaGly), 3.74 (s, 3H, HMeOMe), 3.65, (t, J = 5.3 Hz), & 3.71-3.68 (m), (2H, HPrpc); 3.45 (t, J = 6.0 Hz) & 3.41-3.38 (m), (2H, HPrpa); 1.66-1.64 (m) & 1.77- 1.74 (m) (2H, HPrpb); 1.48 (s) & 1.42 (s), (9H, HMeBoc); HRMS m/z Calcd for C11H21NO5Na 270.1317, Found 270.1320.

Synthesis of (S)-benzyl 2-((3-((tert-butyloxycarbonyl)(2-methoxy-2-oxoethyl) amino) propyl) (methoxycarbonyl)amino)propanoate :

To a cold (0°C) stirring mixture of Ns-Gly-OBn (600 mg, 1.71 mmol) and triphenylphosphine (PPh3) (672 mg, 2.56 mmol) in dry THF (5 ml) under N2 atmosphere was added N-Boc-(3-hydroxympropyl) benzylglycinate (5a) (654 mg, 2.56 mmol) dissolved in THF followed by diisopropyl azodicarboxylate (DIAD) (506 µl, 2.56 mmol). After 15 minutes the ice bath was removed, and the mixture was continued to stir at ambient conditions until TLC indicated the complete consumption of the starting material. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (15 mL) and washed with water (2 X 5 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to denosylation reaction by the addition of thiophenol (210 µl, 2.06 mmol) and K2CO3 (374 mg, 2.72 mmol) in acetonitrile (8 ml). The mixture was continued to stir at ambient conditions until TLC indicated the complete consumption of the starting material. The solvent was removed followed by acidification using 1N HCl (10 ml). The aqueous mixture was washed with diethyl ether to remove all the water-insoluble organic impurities.

The aqueous solution was then treated with K2CO3 until the medium became basic (pH – 10.0). This was followed by the addition of methyl chloroformate (MCF) (262 µl, 3.4 mmol) to the reaction mixture. The mixture was continued to stir at ambient conditions until TLC indicated the complete consumption of the starting material. Removal of solvent resulted in a residue which was dissolved in EtOAc (15 ml), washed with water (2 X 5 ml) and the organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography (EtOAc:Hexane -1:5) to yield the desired product as a viscous oil (530 mg, 1.17 mmol, 88% yield); (TLC-EtOAc:Hexane (1:1) –Rf= 0.4); ¹H NMR (400 MHz, CDCl3, 10 mM) d ppm: (mixture of four rotamers) 7.40-7.30 (m, 5H, HAroBn), 5.16 (s, 2H, HCH2Bn), 4.055 (d, J = 4.08 Hz, 1H, HaGly), 3.99 (d, J = 11.65 Hz, 1H, HaGly), 3.93 (s, 1H, HaGly), 3.84 (d, J = 13.15, 1H, HaGly), 3.75 – 3.70 (m), 3.64 – 3.59 (m) (HMeOMe & HMeMoc), 3.36 – 3.25 (m, 4H, HPrpc & HPrpa), 1.77 – 1.71 (m, 2H, HPrpb), 1.45 (s) & 1.40 (s) (9H, HMeBoc); 13C NMR (100 MHz, CDCl3, 60 mM) d ppm: 171.9, 147.7, 134.3, 133.6, 132.9, 130.5, 125.6, 52.5, 52.47, 19.7; HRMS m/z Calculated for C23H34N2O8K 491.1796, Found 491.1796.

Synthesis of methyl N2-((S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-N6-(tert-butoxycarbonyl)lysinate:
To a cold (-15 °C) stirring solution of Cbz-Glu(OtBu)-OH (3 g , 10 mol) in dry tetrahydrofuran (THF) (30 ml) was added N-methyl morpholine (NMM) (1.64 ml, 15 mol) and ethyl chloroformate (ECF) (1.01 ml, 10.5 mol) and stirred until TLC indicated that all the acid had been consumed to form corresponding mixed anhydride (4 min). To this was added a solution of H2N-Lys(Boc)-OMe (1.67 g, 12.02 mol) in dry THF (10.0 ml) and NMM (2.73 ml, 25 mol). After 2 h the ice-salt bath was removed, and the mixture was stirred at ambient conditions for further 5 h. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (15 mL), washed with water (2 X 5 mL), 1N HCl (2 X 5 mL) and saturated NaHCO3 (2 X 5 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography to yield the desired product (59% yield); HRMS m/z Calculated for C29H45N3O9Na 602.3053, Found 602.3053.

Synthesis of benzyl (5S,8S)-5-(3-(tert-butoxy)-3-oxopropyl)-8-(4-((tert-butoxycarbonyl)amino)butyl)-10-(2-methoxy-2-oxoethyl)-14-(methoxycarbonyl)-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10,14-tetraazahexadecan-16-oate: To a cold (0 °C) solution of 8a (500 mg, 1.07 mmol) in DCM (3.6 ml) was added trifluoroacetic acid (TFA) (715 µl) drop-wise and the mixture was allowed to stir for 2 hrs, by which time TLC indicated the complete consumption of the starting material. Removal of solvent resulted in the Boc-deprotected product 9a as a liquid (515 mg, 1.07 mmol) which was used for further reaction as it is.

To a cold (-15 °C) solution of Cbz-E(OtBu)-K(Boc)-OH (476 mg, 1.94 mmol) in dry tetrahydrofuran (THF) (10 ml) and N-methyl morpholine (NMM) (175 µl, 2.43 mmol) was added ethyl chloroformate (ECF) (112 µl, 1.78 mmol). The mixture was stirred for (4 min) when TLC indicated that all the acid was consumed to form the corresponding mixed anhydride. To this mixture was added a solution of 9a (772 mg, 1.6 mmol) in dry tetrahydrofuran (THF) (4.0 ml) followed by NMM (291 µl, 4.05 mmol). After 2 h of vigorous stirring, the mixture was warmed and stirred until TLC indicated the complete consumption of starting material. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (20 mL), washed with water (2 X 5 mL) and 1N HCl (2 X 5 mL) and saturated NaHCO3 (2 X 5 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography to yield the desired product (308 mg, 0.43 mmol, 65% yield); HRMS m/z Calculated for C45H65N5O14Na 922.4426, Found 922.4426.

Synthesis of HBS-constrained cyclic N-carbamyl single a-helical turn analogue:

MeOH (1 ml) was added to a mixture of 10a (100 mg, 0.14 mmol) and Pd-C (10 mg, 0.1 mol%) in a sealed round-bottomed flask kept under H2-atmosphere and stirred for 30 mins by which time TLC indicated the complete consumption of starting material. Filtering the mixture through Whatman-40 filter paper and concentration of the organic filtrate gave the N- and C-terminal double de-protected derivative of 10a as a viscous liquid 11a (68.8 mg, 0.14 mmol). Next 11a was dissolved in dry acetonitrile followed by addition of EDC (133 mg, 0.57 mmol), HOBT (94 mg, 0.43 mmol) and DIPEA (121 µl, 0.71 mmol) under N2 atmosphere. The mixture was stirred for further 45 hrs. Removal of solvent resulted in a residue which was dissolved in dichloromethane (DCM) (10 mL) and washed with water (2 X 5 mL), 1N HCl (2 X 5 mL) and saturated NaHCO3 (2 X 10 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography to yield the desired product (25 mg, 0.05 mmol, 42% yield).

Characterization of the HBS-constrained cyclic N-carbamyl single a-helical turn analogue Moc-[GE(O-tBu)K(Boc)]G-OMe (1)
Figure 1, panels A and B, show the 2D TOCSY spectra (400 MHz, 10 mM, 10% D2O in H2O, 298 K) solved for Moc-[GE(O-tBu)K(Boc)]G-OMe (1) to assign all the individual spin systems.

Figure 2 shows a Heteronuclear Single Quantum Coherence (HSQC) spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe (1) (400 MHz, 10 mM, 10 % D2O in water , 298 K).

Figure 3 shows the 1H-NMR spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe (1) at 10 mM, D2O, 400 MHz.

Figure 4 shows the 13C-NMR spectrum of HBS-constrained single alpha-helical turn mimic Moc-[GE(O-tBu)K(Boc)]G-OMe at 60 mM, D2O, 400 MHz.

Circular dichroism (CD) studies of the HBS-constrained single a-helical turn template Moc-[GE(OtBu)K(Boc)]G-OMe
The HBS-constrained single a-helical turn helix-nucleating template Moc-[GE(OtBu)K(Boc)]G-OMe shows the CD signature for a classical a-helical turn structure, in the far UV wavelength range (Figure 5). This signature is as follows: there is a positive maximum at ~194 nm and a negative maximum at ~208 nm – both for the p?p* transitions; and a negative maximum at ~224 nm for the n?p* transition. The ratio of ?MRE (n?p*) / ?MRE (p?p*) is ~ 1.00 ± 0.05, indicating a predominantly a-helical turn rather than the competing 310-helicao turn in the a-helicomimic template.

The template shows among the highest known helicities (?MRE (n?p*) ~29 ± 1 deg cm² dmol¯¹) (Figure 5, panel a) in literature for a single helical turn in 10 mM, Tris Cl pH 7.4 buffer at 100 µM helicomimic concentration. The helicity remains almost unperturbed by the addition of the helix inducing solvent TFE (10% in buffer) – which causes <10% increase in helicity values. Thus, the extent of helicity is already high in the HBS-a-helicomimic turn template under aqueous conditions.

Figure 5, panel a) shows the temperature-dependent (278-353 K) CD spectra of Moc-[GE(O-tBu)K(Boc)]G-OMe in 10 mM Tris-chloride aqueous buffer (pH = 7.4), panel b) shows the temperature-dependent (278-353 K) CD spectra of Moc-[GE(O-tBu)K(Boc)]G-OMe in 10% TFE in 10 mM Tris-chloride aqueous buffer (pH = 7.4). The [?MRE]n?p* vs T (K) and [?MRE]p?p* vs T (K) plots of Moc-[GE(O-tBu)K(Boc)]G-OMe showing the nonvariant nature of both the n?p* and p?p* transitions with temperature and hence the remarkable thermal stabilities of the alpha-helical structures in the entire region of the temperature (278 – 353 K) are shown on the right side of each of the CD spectrum.

Since CD ?MRE values primarily report on the excitonic coupling of amide chromophores and is largely independent of the nature of the side chain functional groups, and since the HBS-a-helicomimic turn template is water soluble even without deprotecting the side chain functional groups, the CD was taken for the protected template. These CD ellipticities are hence indicative of the helicities of the backbone helical turn in the template.

Thermal perturbation studies probed by CD spectrometry in the Far-UV wavelength region showed that the HBS-constrained single a-helical turn helix-nucleating template Moc-[GE(OtBu)K(Boc)]G-OMe is highly thermally stable, based on the negligible thermal variation in its ?MRE value upon increasing the temperature by >60 deg C in the buffer. Ther thermal coefficient of ellipticity (??MRE/?T) is <-20 deg cm² dmol¯¹ K¯¹, which is among the lowest known in literature.

The thermal perturbation CD data also shows an isodichroic point at ~200 nm (?), showing the presence of a 2-state equilibrium process operating in the helicomimic. The ??MRE vs T (K) plot also shows this 2-state equilibrium clearly.

Part 2. Synthesis of HBS-constrained extended helicomimic

Scheme 2. List of extended helicomimics (and acyclic analogue) synthesized

Characterization of the synthesized mimics (ERG-A, ERG-B, and ERG-C)
Figure 6, panel A shows the MALDI spectra of helicomimic ERG-A (Moc-[GEK]G-SRA-LRYYY-DK-NH2) (SEQ ID NO: 6). Figure 6, panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Figure 7, panel A shows the MALDI spectra of helicomimic ERG-B (Moc-[GEK]G-ERA-LRYYY-DK-NH2) (SEQ ID NO: 5). Figure 7, panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Figure 8 shows the MALDI spectra of helicomimic ERG-C (Moc-[GEK]G-SRA-ERYYY-DK-NH2) (SEQ ID NO: 4).

Figure 9, panel A shows the MALDI spectra of the acyclic analogue ERG-D (Ac-YDKLSRALRYYYDKN-NH2) (SEQ ID NO: 15). Figure 9, panel B shows the corresponding HPLC chromatogram indicating purity of the compound.

Circular dichroism studies of the helicomimics extended from the HBS-constrained a-helix nucleating template
Figure 10, panel a) shows the temperature-dependent (278-358 K) CD spectra of ERG-A (Moc-[GEK]G-SRA-LRYYY-DK-NH2) (SEQ ID NO: 6), in 10 mM Tris-chloride aqueous buffer (pH = 7.4), Figure 10, panel b) shows the temperature-dependent (278-353 K) CD spectra of ERG-B (Moc-[GEK]G-ERA-LRYYY-DK-NH2) (SEQ ID NO: 5), in 10 mM Tris-chloride aqueous buffer (pH = 7.4), Figure 10, panel c) shows the temperature-dependent (278-353 K) CD spectra of ERG-C (Moc-[GEK]G-SRA-ERYYY-DK-NH2) (SEQ ID NO: 4), in 10 mM Tris-chloride aqueous buffer (pH = 7.4). The [?MRE]n?p* vs T (K) and [?MRE]p?p* vs T (K) plots of ERG-A, ERG-B, ERG-C showing the nonvariant nature of both the n?p* and p?p* transitions with temperature and hence the remarkable thermal stabilities of the alpha-helical structures in the entire region of the temperature are shown on the right side of each of the CD spectrum.

The extended helicomimics ERG-A, ERG-B, and ERG-C, consistently show a positive maximum at ~194 nm, a negative maximum at ~197-198 nm and a negative maximum at ~218-222 nm of wavelength (?). This pattern is indicative of a combination of 2 predominant structures in equilibrium with each other: i) an a-helical structure (positive at ~194 nm, negative at ~208 nm, negative at ~222 nm), mixed with a ß-turn structure (negative at ~194 nm and positive at ~206 nm); and ii) an ensemble of random coil structures (negative at 198 nm). Due to this combination, the negative maxima at 208 nm and 222 nm are not only blue shifted to ~198 nm and to ~218 nm, but its ellipticity is also significantly suppressed relative to that for a canonical, homogeneous a-helical turn.

The thermal perturbation CD data reveal that temperature affects the p?p* transition more than the n?p* transition values. This is largely indicative of an effect of the change in solvent polarity, rather than change in helicomimic helicity, upon heating. Hence, these peptides are quite thermally stable. There is an isodichroic point at ~210 nm. Showing a 2-state transition in the propagating sequence as well, just as in the nucleating template.

No CD signature of any ordered structure is observed for the acyclic negative control ERG-D – indicating the lack of any ordered structure in it. Hence, it is due to the presence of the HBS-constrained single helical turn helicomimic alone, that these helices gain such a-helical order and its transition into a 2-state conformational equilibrium.

Comparing ‘ERG-B’ and ‘ERG-A’:
After closer analysis, the CD of ‘B’ shows a slight improvement in a-helicity as indicated by more negative values for both its n?p* and p?p*transitions, compared to its parent wild type helicomimic (‘A’). So, the observed improvement in the Kd values for ‘B’, compared to ‘A’, is primarily due to subtle changes in the a-helicity induced in ‘B’ by the S366E mutation.

Comparing ‘ERG-C’ and ‘ERG-A’:
The CD of ‘C’ and ‘A’ are very similar, showing that the L369E mutation does not perturb or scramble the helicity by much. So, two other factors could be responsible for the observed remarkably improvement in Kd values of ‘C’ compared to ‘A’. First is that analysis of the mutated co-crystal structure reveals the possible formation of an additional interaction being made by the Glu369 side chain with the underlined, bold, bases in the DNA strand, GAGGA. Second is the improvement in solubility of ‘C’ compared to ‘A’, due to the E369 mutation that has caused the remarkable change in Kd of ‘C’ compared to ‘A’.

The data demonstrates that not only does the HBS-template nucleate and propagate helical order in the appended peptides, but also show that using single point mutations in the propagating peptides, subtle changes in helicities, solubilities, intermolecular interactions, etc. can be introduced into the extended helicomimics. In addition, most importantly, these changes directly attenuate the binding of the HBS-helicomimics with their dsDNA targets.

Figure 11 shows UV-Vis Absorbance vs wavelength plots of ERG-ds-DNA titration with: Panel a) ERG-A, Panel b) ERG-B, Panel c) ERG-C, Panel d) ERG-D, in 10 mM Tris-chloride aqueous buffer (pH = 7.4). Plots of A0/A- A0 (where A0 and A are the absorbances of the DNA and its complex with peptide, respectively) vs 1/[peptide] plots of ERG-A, ERG-B, ERG-C and ERG-D are shown on the right side of each spectrum.

Figure 12 shows Fluorescence Intensity vs wavelength plots of ERG-FAM-ds-DNA titration with: Panel a) ERG-A, Panel b) ERG-B, Panel c) ERG-C, Panel d) ERG-D, in 10 mM Tris-chloride aqueous buffer (pH = 7.4). Plots of 1/F0-F (where Fo-F is the difference in the fluorescence intensity of FAM tagged ds-DNA in native state and in complex with peptide) vs 1/[peptide] plots of ERG-A, ERG-B, ERG-C and ERG-D are shown on the right side of each spectrum.

Figure 13 shows plots of Microscale Thermophoresis (MST) showing fraction of Fluorescently-tagged ds-DNA bond with peptide (ERG-A, ERG-B, ERG-C, ERG-D) vs peptide concentration.

Figure 14 shows plots of Isothermal Titration Calorimetry (ITC) showing kcal mol-1 of injectant vs [peptide/DNA] of ERG-A, ERG-B, ERG-C, ERG-D.

Example 3: Analysis of binding affinities of the peptide mimics
Calculation of Kd Values:
Calculation of Kd from UV-Vis Spectroscopy:
Based upon the variation in absorbance, the intrinsic binding constant/association constant (K) of the drug with DNA can be determined according to Benesi–Hildebrand equation:
A0/ A -A0 = a/(a-b) + a/(a-b) Ka[peptide]
where Ka is the association/binding constant, A0 and A are the absorbances of the DNA and its complex with peptide, respectively, and a and b are the absorption coefficients of the DNA and the DNA-peptide complex.
Calculation of Kd from Fluorescence Spectroscopy:
(F-Fo)/Fo = (F-Fo)/(Fo . I max) + (F-Fo)/(Fo . Kd . Imax . [Peptide])

Kd is calculated from the slope/intercept value where the F-Fo is the change in the fluorescence of FAM tagged ds-DNA in bound and Fo . Imax is the maximum change in the intensity in fluorescence.

Calculation of Kd from Microscale Thermophoresis (MST):
Sample Preparation:
Concentrations of protein and peptide samples were determined using Nanodrop 2000c (ThermoScientific).

Direct Binding Experiments:
Experiments were performed in triplicate in a 96-well plate format. The peptide mimic was concentrated in 10% glycerol in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) using Amicon Ultra Centrifugal filters according to the manufacturer’s protocol. Pluronic F-127 (Sigma) was added to a final concentration of 0.1% in each experiment. Fluorescently-tagged ds-DNA were dissolved in the same buffer as the peptide and were at a final concentration of 35 nM. Each plate incubated for 60 minutes at room temperature and was read on Detector at 25 °C with excitation of 495 nm and emission of 505 nm. Raw values were fit to a Sigmoidal, 4PL, model where X is concentration in GraphPad Prism 8.0. to obtain the EC50 value. The binding affinities (Kd) for each peptide was determined using the following equation as performed in Lao, et al. The following equation was used:
Kd = (RT x (1-FSB) + LST x FSB2)/FSB - LST (1)
RT = EC50 value determined by prism
FSB = fraction bound = 0.5 (in the case of EC50)
LST = total concentration of Flu-ds-DNA = 35 nM (for all experiments).

Calculation of Kd from Isothermal Titration Calorimetry (ITC):
The ITC instrument detects heat that is released or absorbed as a result of the DNA-peptide interaction. This is done by measuring the changes in the power needed to maintain isothermal conditions between the reference and the sample cell. Injections are performed repeatedly, and result in peaks that become smaller as the DNA becomes saturated. Eventually, the peak sizes remain constant and represent only the heats of dilution. Once titration is completed, the individual peaks are integrated by the instrument software. An appropriate binding model is chosen and the isotherm is fitted to yield the Kd value.

Dissociation constants from different methods are tabulated as follows:
The Kd values calculated from UV-Vis, Fluorescence, Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC) are listed in Table 2:

Table 2:

Explanation of an anomalous Kd values for ‘ERG-B’, determined by the Microscale Thermolysis (MST):
MST determines the Kd values in a truly non-invasive way, wherein there is no chemical modification made to the peptide or the dsDNA. Thermolysis depends on molecule/solvent interactions at their interface. Hence, it probes the change in size, charges and solvation entropy of the molecules, compared to their complexes. In B & C, the binding event causes differential burial of the side chain COOH groups of the two different Glu residues. This would affect solvation primarily, compared to the sizes and changes of B & C (which should be similar, since one E is introduced in place of anon-ionizable residues in each case). So, the MS results highlight the effect of the differential solvation of ‘B’ and ‘C’ and perhaps also of their corresponding complexes with the ds-DNA sequence.

These data show the strength of the HBS-constraining method employed in the present disclosure for helicomimic design from native sequences, aimed at not only exactly replicating the biorelevant structure using the native sequences, but also in containing several control elements that can be subtly perturbed in order to significantly attenuate ds-DNA binding, and hence be used as tools for lead-optimization and improving binding and selectivity.

Selectivity Studies:
To probe the dsDNA sequence selectivity, binding studies were performed using the non-invasive UV-vis spectroscopic method, on two helicomimics. The helicomimic ‘A’ has the HBS constraining the native sequence and the helicomimic ‘C’ has the HBS constraining the L?E mutation. ‘C’ shows the best binding constant values among the mutants studied. The ds-DNA sequences were systematically mutated by making a purine?pyrimidine or a pyrimidine?purine mutation at each of the nucleotides in the sequence where crystal structure showed direct interaction with the ERG transcription factor Helix-4.

The Kd values of ERG-A with wild type and randomized ERG-ds-DNA as calculated from UV-Vis spectroscopy are listed in Table 3:
Table 3:
The Kd values of ERG-C with wild type and randomized ERG-ds-DNA as calculated from UV-Vis spectroscopy are listed in Table 4:

Table 4:

Comparison of the Kd values of the complexes containing the wild type dsDNA, with those of the mutated ds-DNA showed that maximum loss in binding was experienced when the underlined, bold, highlighted nucleotides were mutated, as follows - GAGGATG. The strongest effects were observed upon mutation of the cyan highlighted G, since the helicomimic binds to both the ‘G’ and its plectonomically interacting partner ‘C’, in the opposite DNA strand. All other interactions are primarily to one of the bases in the base pairs. These data show a strong selectivity of the HBS-helicomimics for the native dsDNA sequence. Loss of binding up to 2.5 orders of magnitude, compared to the native sequence (constrained by HBS) was observed upon mutation of dsDNA. At the same time, gain in >3 orders of magnitude is observed for binding, between the unconstrained peptide and the HBS-constrained peptide.

Example 4: Synthesis and Characterization of C-terminal extended NLS and TAT containing Cyclic peptides
ERG-H-FNT-001 Moc-[GEK]G-SRA-ERYYY-DK-K(Rh-B)-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 15 (SEQ ID NO: 34)
ERG-H-FNT-002 Moc-[GEK]G-ERA-LRYYY-DK-K(Rh-B)-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 16 (SEQ ID NO: 35)

Table 5

Scheme 3

The synthesis of ERG-H-FNT-001 (SEQ ID NO: 34) and ERG-H-FNT-002 (SEQ ID NO: 35) cyclic analogues was conducted by employing standard conventional Fmoc solid-phase chemistry on Rink-amide resin. The Fmoc deprotection was performed by treating the resin with 20% piperidine in DMF for 5 minutes, followed by washing with DMF 2-3 times. The deprotection step was repeated by immersing the resin in 20% piperidine in DMF for 15 minutes, followed by extensive washing with DMF 5-6 times until the piperidine odour was completely removed.

Each amino acid coupling step was carried out by employing 0.1M Fmoc-protected amino acid, activated by 0.1M Hydroxybenzotriazole (HOBt) and 0.1M N,N’-Diisopropylcarbodiimide (DIPC) in dimethylformamide (DMF) for 3 hours. After all amino acids were coupled, the cyclic molecule Moc-[GE(O-tBu)K(Boc)]G-OH was coupled using 0.1M Moc-[GE(O-tBu)K(Boc)]G-OH, activated by 0.1M Hydroxybenzotriazole (HOBt) and 0.1M N,N’-Diisopropylcarbodiimide (DIPC) in dimethylformamide (DMF) for 3 hours.

The alloc-group was removed by treating the protected resin with tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] in a solution mixture of CHCl3:AcOH:NMM (37:2:1). The subsequent rhodamine coupling was performed using 0.1M Rhodamine B, activated by 0.1M Hydroxybenzotriazole (HOBt) and 0.1M N,N’-Diisopropylcarbodiimide (DIPC) in dimethylformamide (DMF) for 3 hours.

All the side chains of the final molecule and the final molecule was cleaved from the resin employing a 95% TFA cocktail (95% TFA: 2.5% TIPS: 2.5% H2O). The solvent was removed by lyophilization, resulting in a white solid after purification by HPLC.

Figure 15 shows the LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-H-FNT-001 (SEQ ID NO: 34).

Figure 16 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-H-FNT-002 (SEQ ID NO: 35).

Synthesis of C-terminal extended NLS and TAT containing Acyclic peptides:

ERG-P-FNT-001 Ac-YDKL-SRA-LRYYY-DK-K(Rh-B)-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 17 (SEQ ID NO: 36)
ERG-RP-FNT-001 Ac-KD-YYYRL-ARS-LKDY-K(Rh-B)-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 18 (SEQ ID NO: 37)
ERG-NC-FNT-001 Ac-YDGL-SGA-LGYYY-DK-K[Rh-B]-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 19 (SEQ ID NO: 38)
ERG-NC-FNT-002) Ac-YDKL-SRA-LRGGG-DK-K[Rh-B]-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 Figure 20 (SEQ ID NO: 39)
ERG-NC-FNT-003 Ac-YDGL-SGA-LGYGY-DK-K[Rh-B]-GG-PKKKRKV-GG-YGRKKRRQRRR-NH2 MALDI Figure 21 (SEQ ID NO: 40)

Table 6

Scheme 4

The synthesis of acyclic analogues - ERG-P-FNT-001(SEQ ID NO: 36); (ERG-RP-FNT-001) (SEQ ID NO: 37); (ERG-NC-FNT-001) (SEQ ID NO: 38); (ERG-NC-FNT-002) (SEQ ID NO: 39); and (ERG-NC-FNT-003) (SEQ ID NO: 40) - was carried out using standard Fmoc solid-phase chemistry on Rink-amide resin. The Fmoc-deprotection was performed by treating the resin with 20% piperidine in DMF for 5 minutes, followed by washing with DMF 2-3 times. The resin was then treated again with 20% piperidine in DMF for 15 minutes, after which it was washed with DMF 5-6 times until the piperidine odour was no longer detectable.

Each amino acid coupling step was conducted using a 0.1M solution of Fmoc-protected amino acid, activated by 0.1M Hydroxybenzotriazole (HOBt) and 0.1M N,N’-Diisopropylcarbodiimide (DIPC) in dimethylformamide (DMF) for 3 hours.

The alloc-group was subsequently removed by treating the protected resin with tetrakis (triphenylphosphine) palladium(0) [Pd(PPh3)4] in a solution mixture of CHCl3:AcOH:NMM = 37:2:1. Rhodamine coupling was then achieved using 0.1M Rhodamine B, activated by 0.1M Hydroxybenzotriazole (HOBt) and 0.1M N,N’-Diisopropylcarbodiimide (DIPC) in DMF for 3 hours. Finally, all the side chains and the final molecule were cleaved from the resin using a 95% TFA cocktail (95% TFA: 2.5% TIPS: 2.5% H2O). The solvent was removed by lyophilization, resulting in a white solid after purification via HPLC.

Figure 17 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-P-FNT-001 (SEQ ID NO: 36).

Figure 18 shows LC-MS spectrum (A) and analytical HPLC chromatogram (B) of ERG-RP-FNT-001 (SEQ ID NO: 37).

Figure 19 shows the analytical data for ERG-NC-FNT-001 (Acetyl-YDGL-SGA-LGYYY-DKK-GG-PKKKRKV-GG-YGRKKRRQRRR), with Panel A showing the MALDI spectra and Panel B showing the corresponding HPLC chromatogram, indicating the compound's purity. The design of this acyclic peptide involves modifying the original ERG-P-FNT-001 sequence at seven residues to minimize electrostatic interactions between the hotspot residues and DNA.

Original sequence: ERG-P-FNT-001: Acetyl-YDKL-SRA-LRYYYDKK-GG-PKKKRKV-GG-YGRKKRRQRRR
Modified sequence: ERG-NC-FNT-001: Acetyl-YDGL-SGA-LGYGY-DKK-GG-PKKKRKV-GG-YGRKKRRQRRR

In Figure 19, panel A, the following fragments are observed:

1) Chemical Formula: C222H341N70O527- Observed Mass: 4819.6229
2) Chemical Formula: C221H330N60O524- Observed Mass: 4656.5045
3) Chemical Formula: C222H324N48O5212+ Observed Mass: 4494.4118
4) Chemical Formula: C220H319N44O5115+ Observed Mass: 4393.3639
*The exact mass of ERG-NC-FNT-001 is 4827.6811.

Figure 20, panel A shows the MALDI spectra for ERG-NC-FNT-002 (Acetyl-YDKL-SRA-LRGGG-DKK-GG-PKKKRKV-GG-YGRKKRRQRRR). Four rhodamine-B (Rh-B) units are condensed into the oligopeptide, resulting in the observed mass of 6465.319 [M + 3H+].
Panel B displays the corresponding HPLC chromatogram, indicating the compound's purity.

The design of this acyclic peptide modifies the original ERG-P-FNT-001 sequence at seven residues to reduce hydrogen bonding interactions between hotspot residues and DNA.

Original sequence: ERG-P-FNT-001: Acetyl-YDKL-SRA-LRYYYDKK-GG-PKKKRKV-GG-YGRKKRRQRRR
Modified sequence: ERG-NC-FNT-002: Acetyl-YDKL-SRA-LRGGG-DKK-GG-PKKKRKV-GG-YGRKKRRQRRR

Figure 21, Panel A shows the MALDI spectra for ERG-NC-FNT-003. The MALDI spectra reveal the presence of two molecular ions: one salted with three rhodamine-B units and the other with four rhodamine-B units, both associated with the basic residues. Figure 21, panel B shows the corresponding HPLC chromatogram, indicating the compound's purity.

The modified sequence alters the original ERG-P-FNT-001 sequence at seven residues to reduce both hydrogen bonding and electrostatic interactions.

Original sequence: ERG-P-FNT-001: Acetyl-YDKL-SRA-LRYYYDKK-GG-PKKKRKV-GG-YGRKKRRQRRR
Modified sequence: ERG-NC-FNT-003: Acetyl-YDGL-SGA-LGYGY-DKK-GG-PKKKRKV-GG-YGRKKRRQRRR ,CLAIMS:1. A peptide having Formula (I):

(I)
wherein,
P is a protecting group;
-CH2-CH2-CH2- is a propyl linker that is connected to the backbone nitrogen atom of X at one end and to the backbone nitrogen atom of the a-amino group of glycine (G) at the other end;
X is an amino acid, the backbone amino group nitrogen (N) of which is covalently connected to a CH2 group of the propyl linker and to the protecting group as shown in the above formula;
X2 is an amino acid selected from glutamic acid and aspartic acid;
X3 is an amino acid selected from lysine, arginine, ornithine, and histidine;
G is glycine, the backbone nitrogen of which is connected to a CH2 group of the propyl linker;
X4 is an amino acid selected from serine, threonine, aspartic acid, glutamic acid, lysine, histidine, alanine, and leucine;
X5 is an amino acid selected from arginine, lysine, ornithine, and histidine;
X6 is an amino acid selected from alanine, valine, leucine, and isoleucine;
X7 is an amino acid selected from glutamic acid, aspartic acid, lysine, serine, alanine, leucine and isoleucine;
X8 is an amino acid selected from arginine and lysine;
X9 is an amino acid selected from tyrosine and phenylalanine;
X10 is an amino acid selected from tyrosine and phenylalanine;
X11 is an amino acid selected from tyrosine and phenylalanine;
Z is an amino acid;
n = 0, 1, 2, or 3;
R is -OR1 or -NR2R3,
R1 is H or alkyl;
each of R2 and R3 is independently H or alkyl;
and
wherein the peptide mimics Helix 4 of the DNA-binding domain of ERG transcription factor.

2. The peptide as claimed in claim 1, wherein X is glycine or alanine.

3. The peptide as claimed in claim 1 or 2, wherein n= 0.

4. The peptide as claimed in any one of claims 1-3, wherein n=1 and Z is represented as X12, wherein X12 is an amino acid selected from aspartic acid and glutamic acid.

5. The peptide as claimed in any one of claims 1-3, wherein n=2 and Z is represented as X12-X13, wherein X12 is an amino acid selected from aspartic acid and glutamic acid and X13 is an amino acid selected from lysine and arginine.

6. The peptide as claimed in any one of claims 1-3, wherein n=3 and Z is represented as X12-X13-X14, wherein X12 is an amino acid selected from aspartic acid and glutamic acid; X13 is an amino acid selected from lysine and arginine; and X14 is selected from any amino acid or asparagine.

7. The peptide as claimed in any one of claims 1-6, wherein the protecting group P is selected from methoxycarbonyl (Moc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), Allyloxycarbonyl (Alloc), fluorenylmethoxycarbonyl (Fmoc), and nosyl (Ns)

8. The peptide as claimed in any one of claims 1-7, wherein the peptide is selected from:

9. The peptide as claimed in any one of claims 1-8, wherein the peptide shows a binding affinity (Kd) of about 50-450 nM to a double stranded DNA sequence recognized and bound by ERG transcription factor.

10. The peptide as claimed in any one of claims 1-9, wherein a tracer dye is covalently attached to the peptide.

11. The peptide as claimed in any one of claims 1-10, wherein the peptide binds to a double stranded DNA sequence comprising 5’-GAGGA-3’ and 3’-CTAC-5’, wherein in the 3’-CTAC-5’ sequence, CT is complementary to the last two nucleotides GA in the 5’-GAGGA-3’ sequence.

12. A conjugate comprising the peptide as claimed in any one of claims 1-11, wherein the peptide is linked to a moiety at the N or C terminus of the peptide.

13. The conjugate as claimed in claim 12, wherein the moiety is selected from an activator, a repressor, or a nuclear localization sequence (NLS).

14. The conjugate as claimed in claim 13, wherein the activator is selected from Isopropyl ß-D-1-thiogalactopyranoside (IPTG), MarA transcriptional activator, XylS protein, MYB activators, MalT Domain III activator, CAP and/or cAMP receptor protein activator.

15. The conjugate as claimed in claim 13, wherein the repressor is selected from Met Repressor, trp repressor, Pti4 repressor, lac operon repressor, and the L-arabinose operon repressor.

16. The conjugate as claimed in claim 13, wherein the NLS comprises a sequence selected from PKKKRKV (SEQ ID NO: 16) and YGRKKRRQRRR (SEQ ID NO: 17).

17. The conjugate as claimed in claim 13, wherein the conjugate is SEQ ID NO: 34 or SEQ ID NO: 35.

18. A pharmaceutical composition comprising the peptide as claimed in any one of claims 1-11 or the conjugate as claimed in any one of claims 12-17 and a pharmaceutically acceptable excipient.