Fluorescent molecular rotors

Abstract

The present invention relates to methods and compositions for detecting an interaction between a protein and a ligand, comprising: (i) binding at least one fluorescent molecular rotor to said ligand or protein; and (ii) detecting a change in fluorescence emitted by said fluorescent molecular rotor after contact of the bound fluorescent molecular rotor with the other of said ligand or protein, thereby detecting an interaction between the ligand and the protein, wherein the fluorescent molecular rotor comprises: a rotating ?-bond; an electron-donating moiety; an electron-accepting moiety; and a ?-conjugated linker.

Claims

1. A chemical compound of formula (i): ##STR00152## wherein R5 is a OH or a linking moiety selected from the group consisting of a single bond; or optionally substituted heteroalkyl, wherein the main chain atoms of said optionally substituted heteroalkyl are optionally interrupted by one or more optionally substituted cyclic groups; wherein R6 is absent or a ligand, and when R5 is OH, R6 is absent.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

(2) FIG. 1a shows a julodine-based fluorescent molecular rotor, 9-(2-carboxy-2-cyanovinyl) julolidine (CCVJ), corresponding to a disclosed formula (v).

(3) FIG. 1b shows an acridine-based fluorescent molecular rotor corresponding to a disclosed formula (i).

(4) FIG. 1c shows a pyrene-based fluorescent molecular rotor corresponding to a disclosed formula (ii).

(5) FIG. 1d shows a thiazole-based fluorescent molecular rotor corresponding to a disclosed formula (iii).

(6) FIG. 1e shows a carbazole-based (2-arm) fluorescent molecular rotor corresponding to a disclosed formula (iv).

(7) FIG. 1f shows a carbazole-based (3-arm) fluorescent molecular rotor corresponding to a disclosed formula (iv).

(8) FIG. 2 shows a schematic diagram for the synthesis of fluorescent molecular rotors conjugated with biotin.

(9) FIGS. 3a to 3c show that fluorescent molecular rotors of the present invention display an increase in fluorescence upon increase in viscosity. % indicate % of glycerol in ethylene glycol. Viscosity of glycerol>ethylene glycol. Experiments were performed with rotors at a concentration of 100 M.

(10) FIG. 3a depicts a viscosity experiment with julolidine-based molecular rotor.

(11) FIG. 3b depicts a viscosity experiment with a pyrene-based fluorescent molecular rotor.

(12) FIG. 3c depicts a viscosity experiment with acridine-based, thiazole orange-based and carbazole-based fluorescent molecular rotors.

(13) FIGS. 4a and 4b show that fluorescent molecular rotors of the present invention display an increase in fluorescence upon binding to Streptavidin protein.

(14) FIG. 4a depicts the emission spectra of a CCVJ molecular rotor bound to biotin with Streptavidin. Fluorescence increase upon addition of streptavidin to biotin conjugated with CCVJ. 40 M of rotor-biotin conjugate was incubated for 15 min with 50 g of streptavidin. Fluorescence emission spectrum was measured using an excitation wavelength (.sub.ex) of 433 nm.

(15) FIG. 4b depicts the emission spectra of a pyrene-based fluorescent molecular rotor bound to biotin with Streptavidin.

(16) FIG. 5 shows a fluorescence displacement assay using a fluorescent molecular rotor of formula (ii) upon binding of p53 protein to DNA. A decrease in fluorescence signal can be observed upon protein binding. DNA0.44 M (0.11 of rotor); p536.17 M (14 of DNA); Fluorescent molecular rotor4 M.

(17) FIG. 6 shows the emission and excitation spectrums of CCVJ-Biotin with Streptavidin, DNA and both Streptavidin and DNA.

(18) FIG. 7 shows the emission spectras of selected rotors with p53 binding sequence duplex DNA (a) rotor concentration constant at 8 uM with varying DNA ratios, b-d) DNA constant at 4 uM, 0.04 uM, 0.1 uM for b, c and d respectively with varying rotor concentration.)

(19) FIG. 8 shows the emission spectras of acridine-based rotor with p53 and its binding sequence duplex DNA.

(20) FIG. 9 shows the emission and excitation spectras of thiazole-based rotor with p53 and its binding sequence duplex DNA.

(21) FIG. 10 shows the emission and excitation spectras of Carbazole-based (2-arm) rotor with p53 and its binding sequence duplex DNA.

(22) FIG. 11 shows that nutlin dependent inhibition of binding between MDM2 (500 nM) and JP1 (250 nM) results in a concentration dependent decrease in fluorescence activity. Error shows S.D. +/ triplicate readings from duplicate binding reactions. Black and red hashed lines denote maximum fluorescence and background fluorescence from rotor-peptide conjugate, respectively.

(23) FIG. 12 shows the fluorescence of Sybr green rotor, CCVJ rotor and pyrene-based rotor. 100 uM of Sybr green, Pyrene or CCVJ rotors were each added to either 5 ug plasmid DNA, 5 ug DNA with 500 mM NaOH, or PBS buffer only. Resulting fluorescence for each reaction mixture were then compared across the respective filters (Sybr 497/520, Pyrene 376/530, Julolidine 433/505). A weak signal was observed for julolidine and was dropped for further experiments.

(24) FIG. 13 shows the time course measurement of 10 uM (top) or 100 uM (bottom) of a pyrene-based rotor with 5 ug plasmid DNA under various conditions, (A) DNA only (B) DNA+40 mM NaOH (C) DNA+120 mM (D) DNA+200 mM NaOH (E) rotor in buffer only (black trace).

(25) FIG. 14 shows the fluorescence of acrydine-based rotor with DNA and with or without NaOH. Measurements at 480/520 using 50 uM Acrydine with increasing concentration of a 450 bp DNA fragment (PetF and R). Red bars show fluorescence after addition of NaOH to reaction mix.

(26) FIG. 15 shows a fluorescent molecular rotor displacement assay. Reference readings for reaction mixes consisting of 50 uM Acrydine with 150 nM of either control or response element DNA was first taken in the absence of p53 protein. p53 protein was then added at varying concentrations (0, 200 or 500 nM) and incubated at r.t. for 60 minutes before a second measurement was taken. Data is presented as percentage change in fluorescence after the addition of p53 protein (Grey and blue bars) normalized to respective reference measurements (100%, Black bars). Inset shows relative sizes of control- and 5RE-DNA at 125 and 130 bp, respectively. Error bar shows S.D. of 2 separate binding reactions. Red hashed-line indicates reference signal before p53 addition at 100%. 30 uL reaction sample contains 5 uL DNA or buffer blank (75 ng/uL in 10 mM Tris pH 8.0), 4 uL of p53 protein or buffer blank (23 uM in 100 mM Tris pH8.0, 7 mM DTT, 20 mM NaCl) in p53 binding buffer (25 mM phosphate pH7.2, 150 mM KCl, 4 mM DTT).

(27) FIG. 16 shows a biotinylated-rotor DNA pulldown. Commercial streptavidin coated plates were incubated with 50 ul of each biotinylated rotor, or free-biotin (20 uM in PBS) for 20 mins at r.t., washed (2PBST, 2PBS) and blocked with 3% BSA/PBS for 30 mins before adding DNA (450 bp PCR fragment in PBS) across a concentration range (30, 300, or 3000 pg) for a further 20 mins at r.t. Wells are then washed again (3PBST, 3PBS) before DNA was eluted with 150 mM NaOH. Eluate was then neutralized (with 150 mM HCl and 50 mM Tris-Cl, pH 7.4) and used for real-time PCR quantification of template DNA. Data shows fold-increase of rotor captured DNA over background binding in free-biotin control wells. Error depicts S.D. of 2 separate binding reactions.

(28) FIG. 17 schematically depicts p53 function in cells. p53 transcribes target proteins to suppress cancer/cellular transformation. (a) Diagram showing p53 peptide (shaded ribbon) binding to MDM3 N-terminal hydrophobic pocket. (b) Ribbon diagram showing amino acid side-chain projections important for critical contacts in MDM2 binding pocket.

(29) FIG. 18 depicts (top left) Nutlin binding to MDM2 pocket. (top right) Western blot showing cellular levels of p53 target proteins (p21, MDM2) in response to Nutlin treatment.

(30) FIG. 19 schematically shows the design rationale of model system. The box I interaction domain between MDM2 and p53 is used and involves the N-terminal domains of both proteins. MDM2 N-term (18-125) is purified and used for binding with rotor conjugated to either JP1 (SEQ ID NO:1) or JP2 (SEQ ID NO:2) peptide sequences.

(31) FIG. 20a shows that the fluorescence of JP1-R increases when bound specifically to recombinant Mdm2 (residues 18-125) (Top) Rotor-peptide conjugates, JP1-R and JP2-R were added individually to indicated concentrations of Mdm2. (Bot) Fluorescence from binding between Mdm2 and JP1-R was ablated with the addition of Nutlin (50 M). Black hashed-line shows the background fluorescence from 50 nM JP1-R only. Error shows averageS.D. (n=2). (C) JP1-R (50 nM) was added to increasing amounts of Mdm2 (residues 18-125), eiF4e, BSA and IgG. Black hashed-line shows background fluorescence from JP1-R only (50 nM). Error shows averageS.D. (n=2)

(32) FIG. 20b shows that the fluorescence of JP1-R increases when bound specifically to recombinant Mdm2 (residues 18-125) JP1-R (50 nM) was added to increasing amounts of Mdm2 (residues 18-125), eiF4e, BSA and IgG. Black hashed-line shows background fluorescence from JP1-R only (50 nM). Error shows averageS.D. (n=2).

(33) FIG. 21 shows the non-linear fit and calculated apparent K.sub.ds of rotor-peptide conjugates to Mdm2. Fluorescence measurements were taken by titrating either JP1-R (top) or JP2-R (bottom), across a concentration range into solutions containing 100 nM of Mdm2 (18-125). Apparent K.sub.d was calculated using a 1:1 non-linear binding fit model described above. Calculated apparent K.sub.d of Mdm2 to JP1-R or JP2-R, was 16.017.52 nM and 3365640.6 nM, respectively.

(34) FIG. 22 shows an increase in non-specific background fluorescence (JP1-R only) when 0.1% Tween-20 detergent (typical concentration used) was added to PBS binding buffer. However because Tween was needed to give a more consistent measurement, it was subsequently used at 0.005%.

(35) FIG. 23 shows that JP1-R is sensitive to known inhibitors of Mdm2-p53 interaction, as shown in displacement assay using inhibitors. Four different inhibitors of p53-Mdm2: Nutlin-3b (K.sub.d 1.5 M), Nutlin racemic (K.sub.d=201.661 nM), sMTide-02 (K.sub.d=34.42 nM) and sMTide-02a (K.sub.d=6.762 nM), were added to reaction mixtures containing 200 nM of Mdm2 and 80 nM of JP1-R, at concentrations up to 800 nM. Values indicate averageS.D. (n=3).

(36) FIG. 24 shows that the JP1-R reporter is sensitive to several known inhibitors of p53-Mdm2 interaction. (A) Titration of known p53-Mdm2 inhibitors onto pre-bound Mdm2-JP1-R reactions (B) Calculated K.sub.ds using JP1-R of compound inhibitors correlated well with previous reports of K.sub.d.

(37) FIG. 25a shows a synthetic scheme for rotor-peptide conjugates.

(38) FIG. 25b depicts the structure of CCVJ rotor conjugated with JP1 peptide (JP1-R) (SEQ ID NO:1).

(39) FIG. 25c depicts the structure of CCVJ rotor conjugated with JP2 peptide (JP2-R) (SEQ ID NO:2).

(40) FIG. 26a shows a fragment library screen and lead validation for p53-MDM2 inhibition.

(41) FIG. 26b shows that ten out of fifteen hits were validated by fluorescence polarization assay, showing ligand-dependent (100 M, 500 M or 1 mM) displacement. Black bars depict control measurements of FAM-labeled 12.1 peptide, DMSO negative control and 1 M Nutlin positive control. Inactive compound 4A1, used at same concentrations, shows negative displacement control. Error shows S.D. of triplicate measurements.

(42) FIG. 26c shows that weaker inhibitor fragments were further assessed through their ability to displace in vitro translated full-length p53 protein bound to recombinant Mdm2-immobilised on cobalt beads. Western blot shows levels of p53 (upper panel) captured in the presence of indicated compounds (500 uM). Control lanes 1 and 2-show negative control (inactive 4A1 fragment) and positive (100 M Nutlin) displacement events, respectively. Lower panel indicates input Mdm2 levels eluted off beads.

(43) FIG. 26d shows the chemical structures of positive lead compounds.

(44) FIG. 27 shows the in-silico model of rotor-peptide conjugates, (A) JP1-R and (B) JP2-R bound to MDM2 pocket. Hydrogen bond interactions of W8 backbone with S12 backbone and side chain are shown in red-dashed lines. Rotor moiety depicted in green.

EXAMPLES

(45) Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

(46) Materials and Methods

(47) HPLC Purification

(48) All HPLC runs were carried out using an Agilent 1260 infinity and C18 semi-prep column. Solvent eluant systems are of water (+0.065% TFA) and acetonitrile (+0.05% TFA), from 5%-65% in 35 mins. Fractions were then dried and dissolved in water/ACN (1:1). Concentration of samples was obtained through NanoDrop 2000C spectrophotometer absorbance.

(49) Expression and Purification of MDM2

(50) Hexahistidine-MDM2 recombinant protein (residues 18-125) was purified. Briefly, cDNA encoding residues 18-125 of MDM2 was cloned into pET19b (Novagen), transformed into E. coli BL21 (DE3) and induced with 1 mM IPTG. Bacterial cells were then disrupted and first purified using a Ni-nitrilotriacetic acid (NTA) column (eluted with a 1M imidazole gradient), followed by cation-exchange chromatography (eluted using a 1M NaCl gradient).

(51) Determination of Dissociation Constant

(52) The dissociation constants for rotor-peptide conjugates binding to MDM2 (18-125) were determined by titrating JP1-R, or JP2-R, against 100 nM of recombinant MDM2 protein, and fitting experimental data (after subtracting background fluorescence of unbound rotor-peptide conjugates) to the following 1:1 binding model (equation 1).sup.2 where [P] is the MDM2 protein concentration, [L.sub.R] is the concentration of the reporter ligand (rotor-peptide conjugate), K.sub.dR is the dissociation constant. f is the fluorescence signal measured, f.sub.0 is the fluorescence of free unbound rotor-peptide conjugate, f.sub.b is the fluorescence of bound JP-R/MDM2 complex.

(53) $\mathrm{Equation}1$$f=f_0+\left(f_b-f_0\right)\frac{\left(K_{\mathrm{dR}}+\left[L_R\right]+\left[P\right]\right)-\sqrt{{\left(K_{\mathrm{dR}}+\left[L_R\right]+\left[P\right]\right)}^2-4\left[L_R\right]\left[P\right]}}{2\left[L_R\right]}$

(54) The determined apparent dissociation constant of JP1-R binding to MDM2 (K.sub.dR=16.017.52) was later used to determine the apparent K.sub.ds, using competitive binding experiments, of several known MDM2-p53 inhibitor molecules (Racemic Nutlin, Nutlin-3b and sMTide-02). Inhibitor molecules were titrated against a fixed concentration of JP1-R (80 nM) and MDM2 (200 nM) in triplicates and the resulting data points were fitted to equations 2-6.sup.2, 3 where [L.sub.c] and [L.sub.R] denote concentrations of competitive ligand (inhibitor molecules) and reporter ligand (JP1-R), respectively, and K.sub.dC is the dissociation constant of the competitive molecule.

(55) $\begin{array}{cc}f=f_0+\left(f_b-f_0\right)\frac{2\sqrt{\left(d^2-3e\right)}\cos \left(\frac{}{3}\right)-d}{3K_{\mathrm{dR}}+2\sqrt{\left(d^2-3e\right)}\cos \left(\frac{}{3}\right)-d}& \mathrm{Equation}2\\ d=K_{\mathrm{dR}}+K_{\mathrm{dC}}+\left[L_C\right]+\left[L_R\right]-\left[P\right]& \mathrm{Equation}3\\ e=\left(\left[L_R\right]-\left[P\right]\right)K_{\mathrm{dR}}+\left(\left[L_C\right]-\left[P\right]\right)K_{\mathrm{dC}}+K_{\mathrm{dR}}{K}_{\mathrm{dC}}& \mathrm{Equation}4\\ g=-K_{\mathrm{dR}}{K}_{\mathrm{dC}}\left[P\right]& \mathrm{Equation}5\\ ={\cos }^{-1}\left[\frac{-2d^3+9\mathrm{de}-27g}{2\sqrt{{\left(d^2-3e\right)}^3}}\right]& \mathrm{Equation}6\end{array}$

(56) Curve fitting and calculations for respective K.sub.d values were performed using the software Prism 5.03 (Graphpad).

(57) Fragment Library Screen with Rotor-Peptide Probe, JP1-R

(58) Unless otherwise stated, binding reactions involving rotor-peptide conjugates were all performed using black 96-well polypropylene plates (Corning), in a PBS reaction buffer solution containing 0.005% Tween-20 (v/v). For reactions involving Zenobia fragment library compounds, 10% DMSO was added to increase solubility of compounds. Fluorescence activity was measured using the EnVision Multilabel reader (Perkin Elmer) at 435/505 (ex/em) nm.

(59) A small-fragment library (Zenobia Therapeutics) was sampled in our compound screen using JP1-R. The library consists of 352 fragment molecules with decorated-ring structures and an average molecular weight of 154.2 Da. The screen was performed by first aliquoting a pre-made mixture containing 80 nM JP1-R and 200 nM MDM2 (18-125) onto a 96-well plate, and allowing it to equilibrate to room temperature (15 min) before a first reference reading was measured. Next, 500 M of each fragment compound was added into the JP1-R/MDM2 reaction mixture, and incubated for another 30 min before a second measurement was taken. The final result was interpreted as a percentage change of the second measurement from the first reference measurement, of which a decrease in fluorescence would indicate a positive hit (from displacing JP1-R off MDM2). The displacement threshold was set, for this study, at 20% reduction from the reference fluorescence value.

(60) Fluorescence Polarization Assay

(61) Fluorescence polarization was performed as previously described, except in a reaction buffer containing PBS, 10% DMSO (v/v), 0.005% Tween-20 (v/v). Briefly, a mixture containing 50 nM of FAM-labeled 12.1 peptide (RFMDYWEGLNK) and 250 nM of recombinant MDM2 was constituted before adding 500 M of respective fragment library compound. The reactions were then incubated at room temperature for 30 min before measuring on the EnVision plate reader (Perkin Elmer).

(62) Pull-Down Assay and Western Blotting

(63) 650 ng of MDM2 (18-125) recombinant protein was first incubated with either 500 M of the respective fragment compound, 100 M Nutlin, or DMSO (in a reaction buffer containing PBS, 10% DMSO (v/v), 0.005% Tween-20 (v/v), 0.2% BSA (w/v) for 15 min at room temperature before adding to 5 L of IVT-translated p53 protein for another 30 min. p53 protein was synthesized using the PURESYSTEM (NEB) in-vitro transcription/translation kit as previously described, but reconstituted with 6 binding buffer (150 mM NaPi, pH 7.2, 600 mM NaCl, 24 mM DTT) upon completion of IVT reaction. To capture the MDM2/p53 complex, pre-blocked (2% BSA/PBS for 2 hours at room temperature) Dynabeads His-Tag Isolation & Pulldown (Life Technologies) beads were added to the compound/MDM2/IVTp53 mixture and rotated at room temperature for 15 min. After which, beads were washed 4 with wash buffer (PBS, 0.1% BSA (w/v), 0.1% Tween-20 (v/v), 5 mM imidazole), once with PBS (15s vortex at 1600 r.p.m. on MS2 minishaker, IKA) and eluted with 25 L LDS at 95 C. for 7 min. Western blotting of the pull-down eluates were performed as previously described, and probed with either DO-1 (anti-p53) or 4B1 (anti-MDM2) primary antibodies.

(64) Molecular Modeling Studies

(65) a) Molecular Dynamics Simulations

(66) The initial structure of MDM2-peptide complex was taken from the crystal structure of high affinity peptide bound to MDM2 (PDB code 3EQS, resolved at 1.7 ). Peptides were modeled using Modeller.sup.7 was used to model the peptides using PMI peptide as template. The structure used included residues 25-109 of the N-terminal domain of human MDM2 and residues 1-13 of the peptides JP1-R (.sup.1MPRFMDYWEGLSK.sup.13-rotor) and JP2-R (.sup.1MPRFMDYWEGLNK.sup.13-rotor). Rotor was modeled in the Xleap module in AMBER; RESP atomic charges for the peptides were derived using the RED server. To keep the ends capped and neutral, the N- and C-termini of MDM2 were capped with acetyl (ACE) and N-methyl (NME) moieties respectively, while the N- and C-termini of the peptide were capped with acetyl (ACE) and amidate (NH.sub.2) respectively. Molecular dynamics simulations were performed with the SANDER module of the AMBER11 package employing the all-atom ff99SB force field. Each system was simulated for 100 ns at constant temperature (300 K) and pressure (1 atm), and structures were stored every 10 ps. Simulations were carried out for the complexes of MDM2 with the rotor analogs JP1-R and JP2-R. DSSP was used to calculate the secondary structures of the peptides. The simulation protocol was the same as reported earlier.

(67) b) Replica Exchange Molecular Dynamics Simulations

(68) We used Replica Exchange Molecular Dynamics (REMD) to investigate the stability of the helicity of the peptides. This REMD approach involves evolving a number of copies of the system in parallel at different temperatures and periodically exchanging the configurations between trajectories. This insures proper canonical sampling at all temperatures, with high-temperature simulations facilitating barrier crossings and low temperature simulations to explore local free energy minima. The systems were prepared and relaxed as described earlier. In each case, 24 replicas of the system were evolved in parallel for 20 ns at constant NVT, with temperatures evenly spaced between 300 and 400K in approximately 4-K intervals. After the first 100 ps, replica exchanges between each pair of nearest neighbor trajectories were attempted every 12 ps to equilibrate the system. The configurations were saved in 1-ps intervals over 20 ns of each simulation, providing an aggregate 480 ns of sampling for reach system with the exchange acceptance range of 36% to 45%.

(69) Viscosity Experiment General Procedure

(70) The rotors were dissolved in spectroscopy-grade DMSO to obtain a concentrated stock solution of 100 mM. The solution was vortexed to ensure complete dissolution.

(71) For each rotor, 6 uL of the 100 mM stock was dissolved in 1200 uL of ethylene glycol in each 1.5 mL tube.

(72) 3 mL of glycerol was then heated in a boiling bath to reduce viscosity and improve pipetting.

(73) In 5 separate tubes, add 200 uL of stained ethylene glycol to 800 uL of unstained ethylene glycol; 200 uL of stained ethylene glycol, 600 uL of unstained ethylene glycol to 200 uL glycerol; 200 uL of stained ethylene glycol, 400 uL of unstained ethylene glycol to 400 uL of glycerol; 200 uL of stained ethylene glycol, 200 uL of unstained ethylene glycol to 600 uL glycerol; and 200 uL of stained ethylene glycol to 800 uL glycerol. The fluids will then have a glycerol content of 0%, 20%, 40%, 60%, 80% respectively.

(74) The five tubes were then placed on an inverting mixer and allowed to mix for at least 2 hours while the temperature equilibrate to room temperature.

(75) Measurements were taken using 96-well Corning Flat-bottom Black plate and Tecan Infinite M-1000 Spectrophotometer.

(76) Experiment with DNA/p53/Rotors. General Procedure.

(77) For the first set with DNA only, 1PBS buffer and p53 6 binding buffer were first added to the wells. Subsequently, rotor was added. Lastly, DNA was added. The combined solutions were mixed in each well and allowed to incubate in the dark at room temperature for, half an hour before data on fluorescence intensity was gathered.

(78) For the second set with p53 only, 1PBS buffer and p53 6 binding buffer were first added to the wells. Subsequently, rotor was added. Lastly, p53 was added. The combined solutions were mixed in each well and allowed to incubate in the dark at room temperature for half an hour before data on fluorescence intensity was gathered.

(79) For the third set with DNA and p53, 1PBS buffer and p53 6 binding buffer were first added to the wells. Subsequently, rotor was added and DNA was added. The combined solutions were mixed in each well and allowed to incubate in the dark at room temperature for half an hour before p53 was added. The combined solutions were then allowed to incubate in the dark at room temperature for another half an hour before data on fluorescence intensity was gathered.

(80) 6 binding buffer consists: 150 mM sodium phosphate buffer, 600 uM KCl, 24 mM DTT.

(81) Measurements were taken using Greiner 384 black flat-clear-bottom for 18 uL mixtures, Corning 96 flat-black half-area for 80 uL and 100 uL mixtures, and Tecan Infinite M-1000 Spectrophotometer.

(82) TABLE-US-00007 TABLE 1 Experimental data for Rotor/DNA/P53 Thiazole- Carbazole- Carbazole- Acridine-based based based (2- based (3- Compound rotor rotor arm) rotor arm) rotor Emission 526 552 572 582 wavelength (nm) Excitation 480 510 458 456 wavelength (nm) 2X 3.33X 9.1X 3.33X 15X 3.33X 15X 3.33X 15X Final rotor 8 8 8 0.13 0.6 4 4 1.5 concentration (uM) Final DNA 4 2.4 0.88 0.04 0.04 1.2 0.27 0.1 concentration (uM) Final p53 16.64 9.98 3.66 0.17 0.17 4.99 1.12 0.42 Concentration (uM) - 4.16X of DNA Total volume 18 100 18 80 (uL)

(83) TABLE-US-00008 TABLE 2 Experimental data for Rotor/DNA Carbazole- Carbazole- Acridine - Thiazole- based (2- based (3- Compound based rotor based rotor arm) rotor arm) rotor Emission 526 552 572 582 wavelength (nm) Excitation 480 510 458 456 wavelength (nm) Final rotor 8 Varies Varies Varies concentration (uM) Final DNA Varies 0.04 4 0.1 concentration (uM) Total volume 100 100 100 80 (uL)

(84) TABLE-US-00009 TABLE 3 Experimental data for Rotor viscosity test Carbazole- Carbazole- Acridine - Thiazole- based (2- based (3- Compound based rotor based rotor arm) rotor arm) rotor Emission 526 552 572 582 wavelength (nm) Excitation 480 510 458 510 (From wavelength Em spectra, (nm) should be 456 nm.) Final rotor 10 100 100 100 concentration (uM) Total volume 100 100 100 100 (uL)
Modeling Results

(85) TABLE-US-00010 TABLE 4 Helical propensity of JP1-R and JP2-R probes in their unbound conformations JP1-R % Helicity JP2-R % Helicity M 0 M 0 P 5 P 1 R 26 R 20 F 44 F 45 M 51 M 52 D 54 D 59 Y 44 Y 49 W 49 W 50 E 42 E 44 G 43 G 35 L 34 L 22 S 12 N 5 K 0 K 0

(86) TABLE-US-00011 TABLE 5 Components of binding free energy (in kcal/mol) of MDM2 with JP1-R and JP2-R peptides MDM2- JP1-R MDM2 JP1-R Delta ELE 2910.6 65.2 2400.8 64.3 355.1 20.8 154.7 VDW 410.6 16.4 304.9 14.7 11.8 5.5 94.0 GAS 1015.6 69.3 761.5 66.9 4.0 23.4 250.2 GBSOL 1348.9 60.5 1249.2 60.4 279.7 20.1 180.1 GBTOT 2364.5 32.6 2010.7 29.1 283.7 12.0 70.1 TSTOT 1244.1 6.5 1056.6 3.8 228.3 5.2 40.8 G.sub.bind 29.3 -MDM2- JP2-R MDM2 JP2-R Delta ELE 2822.7 68.3 2383.4 59.7 288.0 30.0 151.3 VDW 409.4 17.3 304.9 14.9 14.9 6.6 89.6 GAS 933.8 72.9 742.8 64.3 51.2 33.1 242.2 GBSOL 1356.1 61.6 1265.3 53.0 266.9 29.6 176.2 GBTOT 2289.9 31.9 2008.2 29.1 215.7 12.0 66.0 TSTOT 1240.5 5.6 1058.3 5.1 225.8 6.2 43.5 G.sub.bind 22.5

(87) TABLE-US-00012 TABLE 6 Residue wise energy contributions (in kcal/mol) of JP1-R peptides for their interactions with MDM2 Residue TVDW TELE TGAS TGB TGBSUR TGBSOL TGBTOT M 0.8 0.1 0.9 0.6 0.1 0.5 0.4 P 0.9 0.5 0.4 0.2 0.1 0.2 0.2 R 1.4 28.9 27.5 27.9 0.1 27.9 0.5 F 7.1 3.9 10.9 4.8 0.8 4.0 6.9 M 3.1 1.8 4.8 2.6 0.4 2.1 2.7 D 0.3 38.7 39.0 39.2 0.0 39.2 0.3 Y 4.0 1.9 5.9 3.7 0.4 3.3 2.6 W 6.9 4.3 11.2 5.9 0.7 5.2 6.0 E 0.6 45.4 46.0 46.4 0.1 46.4 0.4 G 0.4 1.1 1.5 1.6 0.0 1.6 0.1 L 4.6 3.8 8.3 4.1 0.5 3.6 4.7 S 3.0 1.6 4.6 3.8 0.3 3.5 1.0 K 2.6 1.0 3.6 1.4 0.2 1.2 2.4 Rotor 4.2 0.6 6.1 0.9 0.5 0.4 5.7

(88) TABLE-US-00013 TABLE 7 Residue wise energy contributions (in kcal/mol) of JP2-R peptides for their interactions with MDM2 Residue TVDW TELE TGAS TGB TGBSUR TGBSOL TGBTOT M 0.7 0.3 1.0 0.9 0.1 0.8 0.2 P 0.6 0.1 0.8 0.6 0.1 0.5 0.3 R 1.2 27.5 26.3 26.9 0.1 27.0 0.7 F 6.8 3.7 10.4 4.8 0.8 4.0 6.4 M 2.9 1.6 4.5 2.4 0.4 2.0 2.5 D 0.2 37.1 37.3 37.5 0.0 37.5 0.2 Y 3.7 3.0 6.6 5.0 0.4 4.6 2.1 W 6.7 4.1 10.8 5.7 0.7 4.9 5.8 E 0.6 44.0 44.5 45.0 0.0 44.9 0.4 G 0.3 1.0 1.3 1.5 0.0 1.5 0.2 L 4.5 1.4 5.9 2.9 0.5 2.4 3.5 N 3.1 3.8 6.9 6.8 0.3 6.5 0.3 K 2.7 0.8 3.5 1.3 0.2 1.0 2.5 Rotor 2.9 0.7 4.8 1.4 0.5 1.8 3.0

(89) TABLE-US-00014 TABLE 8 Calculated K.sub.ds using JP1-R of compound inhibitors correlated well with previous reports of K.sub.d (FIG. 24). Apparent K.sub.d Compound Previous reported K.sub.d (nM) using JP.sub.1-R (nM) Racemic Nutlin 201.61 60.85 (ITC) 164.8 29.43 sMTide-02 34.35 2.03 (FP) 13.01 4.29 Nutlin-3b N.A. 2461 687.3

(90) TABLE-US-00015 TABLE 9 Amino acid sequence and dissociation constants of disclosed peptide variants Peptide Aminoacid ID sequence K.sub.d.sup.# JP1 MPRFMDYWEGLSK 18.83 5.03 (SEQ ID NO: 1) JP2 MPRFMDYWEGLNK 239.81 53.79 (SEQ ID NO: 2) .sup.#Without lysine residue at C-terminus

Example 1a: Synthesis of Acridine-Based Fluorescent Molecular Rotor

(91) ##STR00136##

(92) Acridine Orange Aldehyde (0.12 g, 0.42 mmol, 1 eq.) and cyanoacetic acid (0.053 g, 0.63 mmol, 1.5 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.29 mL, 2.09 mmol) was then added to the reaction mixture after solvating in anhydrous DMF. The reaction mixture was heated to 55 C. overnight, then evaporated to dryness and purified via column chromatography to yield orange solids (1.69 mg, 1%).

(93) .sup.1H NMR (CDCl.sub.3, 400 MHz) 1.26 (s, 3H), 3.05 (s, 3H), 3.24 (s, 6H), 7.06 (dd, 1H, J=2.4, 9.3 Hz), 7.31 (s, 1H), 7.47 (s, 1H), 7.60 (d, 1H, J=9.0 Hz), 7.69 (d, 1H, J=9.4 Hz), 8.36 (s, 1H). .sup.13C NMR (100 MHz, CDCl.sub.3) 154.8, 154.6, 143.7, 142.8, 141.5, 130.4, 130.3, 117.8, 117.2, 115.2, 95.4, 93.8, 40.6, 30.4, 29.9.

Example 1b: Synthesis of Acridine Rotor-Biotin

(94) ##STR00137##

(95) Acridine Orange aldehyde (0.0190 g, 0.065 mmol, 1.0 eq.) and Biotin-CN (0.023 g, 0.065 mmol, 1.0 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.065 mL, 0.47 mmol, 7.0 eq.) was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 55 C. overnight, then evaporated to dryness and purified via column chromatography to yield pale orange solids.

(96) .sup.1H NMR (CDCl.sub.3, 400 MHz) 0.87 (m, 1H), 1.24 (m, 3H), 1.54 (m, 2H), 2.70 (m, 2H), 2.91 (s, 6H), 2.98 (s, 6H), 3.02 (d, 2H, J=5.0 Hz), 3.23 (d, 3H, J=3.4 Hz), 3.29 (m, 1H), 4.26 (t, 1H, J=6.8 Hz), 6.77 (d, 1H, J=2.4 Hz), 7.01 (d, 1H, J=8.3 Hz), 7.18 (d, 1H, J=2.4 Hz), 7.34 (d, 1H, J=8.6 Hz), 7.49 (d, 1H, J=8.3 Hz), 8.06 (dd, 1H, J 9.5, 1.4 Hz), 8.26 (d, 1H, J=9.6 Hz). .sup.13C NMR (100 MHz, CDCl.sub.3) 171.8, 155.1, 152.3, 151.0, 139.4, 136.8, 134.5, 131.4, 130.8, 120.2, 117.5, 117.2, 115.4, 115.3, 114.9, 113.7, 101.9, 95.3, 77.4, 56.8, 46.0, 45.4, 40.6, 38.4, 36.7, 29.8, 27.9, 24.9, 20.4, 16.2. HRMS (TOF MS ES+): calcd for C.sub.33H.sub.39KN.sub.7O.sub.4S [M+K].sup.+ 668.2421. found 668.9434.

Example 2: Synthesis of Thiazole-Based Fluorescent Molecular Rotor

(97) ##STR00138##

Preparation of 3-methyl-2-(methylthio) benzo[d]thiazol-3-ium 4-methylbenzenesulfonate

(98) 2-methylthiobenzothiazole (5.00 g, 27.62 mmol, 1 eq.) was weighed into a 25 mL round bottom flask under argon. Methyl p-toluenesulfonate (4.58 mL, 30.39 mmol, 1.1 eq.) was then added and the reaction mixture was allowed to stir at 130 C. for 1 hour. Acetone was then added after cooling to 70 C. until white precipitate appeared. The mixture was then refluxed for another 30 minutes before cooling to room temperature. Precipitate was collected by filtration and dried to yield pale yellow solids (10.02 g, 99%).

(99) .sup.1H NMR (CH.sub.3OD, 400 MHz) 2.35 (s, 3H), 3.12 (s, 3H), 4.15 (s, 3H), 7.19 (d, 2H, J=4.0 Hz), 7.67 (d, 2H, J=8.0 Hz), 7.73 (t, 1H, j=8.0 Hz), 7.85 (t, 1H, J=8.0 Hz), 8.07 (d, 1H, J=8.0 Hz), 8.22 (d, 1H, J=8.0 Hz). .sup.13C NMR (CH.sub.3OD, 100 MHz) 183.3, 144.2, 143.7, 141.6, 130.8, 130.0, 129.8, 128.6, 127.0, 124.7, 116.5, 36.9, 21.3, 18.5.

(100) ##STR00139##

Preparation of 1-(3-aminopropyl)-4-methylquinolin-1-ium bromide

(101) 3-bromopropylamine hydrobromide (6.06 g, 27.68 mmol, 1.46 eq.) was weighed into an argon-flushed 25 mL round-bottom flask. Ethanol (5 mL) was then added to dissolve. Upon addition of Lepidine (2.5 mL, 18.91 mmol, 1 eq.), the reaction mixture was heated to 40 C. overnight. Pale pink precipitate (1.67 g, 32%) formed was filtered, washed and dried.

(102) .sup.1H NMR (D.sub.2O, 400 MHz) 2.55 (quin, 2H, J=8.0 Hz), 3.10 (s, 3H), 3.29 (t, 2H, J=8.0 Hz), 5.16 (t, 2H, J=8.0 Hz), 7.98 (d, 1H, J=4.0 Hz), 8.11 (t, 1H, J=4.0 Hz), 8.32 (t, 1H, J=8.0 Hz), 8.46 (d, 1H, J=8.0 Hz), 8.60 (d, 1H, J=8.0 Hz). .sup.13C NMR (100 MHz, D.sub.2O) 160.6, 147.3, 137.2, 135.6, 129.8, 127.3, 122.6, 118.2, 54.3, 36.5, 27.0 19.6.

(103) ##STR00140##

(104) Preparation of Thiazole Orange Scaffold.

(105) 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium 4-methylbenzenesulfonate (1.05 g, 2.86 mmol, 1 eq.) and 1-(3-aminopropyl)-4-methylquinolin-1-ium bromide (0.8 g, 2.86 mmol, 0.1 eq.) was weighed into 25 mL round-bottom flask under argon. 30 mL of ethanol was added to dissolve. Triethylamine (0.8 mL) was then added and the reaction was stirred at room temperature for 1 hour. Red precipitate (0.16 g, 11%) formed upon addition of ether was filtered, washed and dried.

(106) .sup.1H NMR (CH.sub.3OD, 400 MHz) 1.21 (t, 1H, J=8.0 Hz), 2.31 (td, 2H, J=Hz), 3.00 (s, 3H), 3.18 (m, 3H), 3.88 (s, 3H), 6.70 (t, 1H, J=8.0 Hz), 6.74 (s, 1H), 6.77 (d, 1H, J=8.0 Hz), 6.90 (td, 1H, J=4.0, 8.0 Hz), 7.14 (d, 1H, J=8.0 Hz), 7.23 (dd, 1H, J=4.0, 8.0 Hz), 7.32 (t, 1H, J=8.0 Hz), 7.50-7.71 (m, 4H), 7.88 (t, 1H, J=8.0 Hz), 8.08 (d, 1H, J=8.0 Hz), 8.29 (d, 1H, J=8.0 Hz), 8.54 (d, 1H, J=8.0 Hz). .sup.13C NMR (100 MHz, CH.sub.3OD) 162.1, 158.4, 150.8, 145.3, 142.2, 141.9, 139.1, 134.3, 129.4, 128.1, 127.5, 126.5, 123.7, 123.3, 122.1, 119.4, 113.6, 110.0, 109.5, 89.1, 55.2, 52.3, 34.0, 30.5, 30.2.

Example 3: Synthesis of Carbozole-Based (2-Arm) Fluorescent Molecular Rotor

(107) ##STR00141##

Preparation of 3,6-dibromo-9-phenyl-9H-carbazole

(108) 9-phenyl-9H-carbazole (0.3 g, 1.23 mmol, 1 eq.) was dissolved in DMF. N-Bromosuccinimide (0.44 g, 2.47 mmol, 2 eq.) was then added slowly and the resultant mixture was allowed to stir at room temperature overnight. The reaction mixture was then poured into brine and extracted with DCM. The organic extracts were then dried with Na.sub.2SO.sub.4 and concentrated. Crude product was then re-precipitated with methanol and THF gave white solids as 3,6-dibromo-9-phenyl-9H-carbazole (0.32 g, 64%).

(109) .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.24 (s, 1H), 7.48-7.52 (m, 5H), 7.59-7.64 (m, 2H), 8.20 (d, 2H, J=2.0 Hz). .sup.13C NMR (100 MHz, CDCl.sub.3) 140.1, 137.0, 130.3, 129.5, 128.3, 127.2, 124.1, 123.4, 113.2, 111.7.

(110) ##STR00142##

Preparation of 9-phenyl-3,6-bis((E)-2-(pyridin-4-yl)vinyl)-9H-carbazole

(111) 3,6-dibromo-9-phenyl-9H-carbazole (96.4 mg, 0.24 mmol, 1 eq.), palladium diacetate (5 mg, 22.5 mol, 0.09 eq.), tris-o-tolylphosphine (12.2 mg, 0.04 mmol, 0.16 eq.) and 4-vinylpyridine (0.17 mL, 1.55 mmol, 6.2 eq.) were added to triethylamine dissolved in degassed THF. The reaction mixture was then heated for 4 days at 110 C. in a sealed tube, and subsequently, diluted in DCM and filtered over celite after cooling to room temperature. The filtrate was washed with brine, dried over Na.sub.2SO.sub.4 and evaporated to dryness to afford a brown paste that was purified by column chromatography to yield yellow solids (80.8 mg, 72%).

(112) .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.10 (d, 2H, J=16.0 Hz), 7.39 (s, 1H), 7.42 (d, 5H, J=4.0 Hz), 7.51-7.59 (m, 6H), 7.63-7.67 (m, 4H), 8.34 (s, 2H), 8.59 (d, 4H, J=4.0 Hz). .sup.13C NMR (100 MHz, CDCl.sub.3) 150.3, 145.3, 141.9, 134.7, 134.0, 130.3, 129.1, 127.2, 125.7, 124.1, 123.9, 123.7, 120.8, 119.5, 110.7. HRMS (TOF MS ES+): calcd for C.sub.32H.sub.23N.sub.3Na [M+Na].sup.+472.1784. found 472.1779.

(113) ##STR00143##

Preparation of 4,4-((1E,1E)-(9-phenyl-9H-carbazole-3,6-diyl)bis(ethene-2,1-diyl))bis(1-methylpyridin-1-ium)

(114) 9-phenyl-3,6-bis((E)-2-(pyridin-4-yl)vinyl)-9H-carbazole (80.8 mg, 0.18 mmol) was weighed into round bottom flask and dissolved in a DCM/MeOH (1:1 v:v) mixture. Methyl iodide (2 mL, 32.13 mmol) was then added. The solution was heated at reflux for 3 days and cooled to room temperature. The red precipitate (111.3 mg, 84%) was filtered and dried.

(115) .sup.1H NMR (DMSO, 400 MHz) 4.27 (s, 6H), 7.50 (d, 2H, J=8.0 Hz), 7.57-7.64 (m, 3H), 7.65-7.78 (m, 4H), 7.93 (d, 2H, J=8.0 Hz), 8.23 (d, 5H, J=8.0 Hz), 8.27 (s, 1H), 8.72 (s, 2H), 8.84 (D, 4H, J=4.0 Hz). .sup.13C NMR (100 MHz, DMSO) 152.8, 144.9, 142.0, 141.5, 135.8, 130.4, 128.5, 128.2, 127.2, 126.8, 123.2, 123.1, 121.2, 110.9, 46.8.

Example 4: Synthesis of Carbozole-Based (3-Arm) Fluorescent Molecular Rotor

(116) ##STR00144##

Preparation of 9-(4-bromophenyl)-9H-carbazole

(117) Copper (II) sulphate (1.35 g, 8.48 mmol, 0.8 eq.), potassium carbonate (2.93 g, 21.20 mmol, 2 eq.), dibromobenzene (5.00 g, 21.20 mmol, 2 eq.) and carbazole (1.77 g, 10.60 mmol, 1 eq.) were heated in a round-bottom flask at 210 C. overnight under argon. After cooling to room temperature, water was added to stop the reaction and the mixture was extracted with DCM. The organic layer was dried with Na.sub.2SO.sub.4, concentrated in vacuo and purified via column chromatography to yield white crystals (1.05 g, 31%).

(118) .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.28-7.59 (m, 9H), 7.74 (d, 1H, J=8.6 Hz), 8.14 (d, 1H, J=7.8 Hz), 8.19 (d, 1H, J=7.8 Hz). .sup.13C NMR (100 MHz, CDCl.sub.3) 140.8, 137.0, 133.3, 128.9, 126.2, 123.7, 121.1, 120.5, 120.4, 109.7. HRMS (TOF MS ES+): calcd for C.sub.18H.sub.12 BrNK [M+K].sup.+ 359.9790. found 359.2444.

(119) ##STR00145##

Preparation of 3,6-dibromo-9-(4-bromophenyl)-9H-carbazole

(120) To a solution of 9-(4-bromophenyl)-9H-carbazole (0.1 g, 0.31 mmol, 1 eq.) in DMF (3.5 mL), N-Bromosuccinimide (0.12 g, 0.69 mmol, 2.2 eq.) in DMF (3.5 mL) was added dropwise and the mixture was allowed to stir at room temperature for 3 hours. Water was then added to give white precipitate which was recrystallized with hexane to yield white solids (0.082 g, 55%).

(121) .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.20 (d, 2H, J=8.5 Hz), 7.37 (d, 2H, J=8.6 Hz), 7.50 (dd, 2H, J=1.9, 8.7 Hz), 7.73 (d, 2H, J=8.5 Hz). .sup.13C NMR (100 MHz, CDCl.sub.3) 139.8, 136.0, 133.5, 129.7, 128.7, 124.2, 123.5, 121.9, 113.5, 111.4. HRMS (TOF MS ES+): calcd for C.sub.18H.sub.10 Br.sub.3N [M].sup.+ 478.8338. found 478.8349.

(122) ##STR00146##

Preparation of 3,6-bis((E)-2-(pyridin-4-yl)vinyl)-9-(4-((E)-2-(pyridin-4-yl)vinyl)phenyl)-9H-carbazole

(123) 3,6-dibromo-9-(4-bromophenyl)-9H-carbazole (0.082 g, 0.17 mmol, 1 eq.), palladium diacetate (4.8 mg, 21.4 mol, 0.13 eq.), tris-o-tolylphosphine (14.2 mg, 21.4 mol, 0.27 eq.) and 4-vinylpyridine (0.17 mL, 1.59 mmol, 9.3 eq.) were added to triethylamine dissolved in degassed THF. The reaction mixture was then heated for 4 days at 110 C. in a sealed tube, and subsequently, diluted in DCM and filtered over celite after cooling to room temperature. The filtrate was washed with brine, dried over Na.sub.2SO.sub.4 and evaporated to dryness to afford a brown paste that was purified by column chromatography to yield orange solids.

(124) .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.08 (t, 5H, J=16.1 Hz), 7.36-7.41 (m, 15H), 7.77 (d, 2H, J=8.4 Hz), 8.31 (s, 2H), 8.54 (d, 6H, J=6.1 Hz).

(125) ##STR00147##

Preparation of 4,4-((1E,1E)-(9-(4-((E)-2-(1-methylpyridin-1-ium-4-yl)vinyl)phenyl)-9H-carbazole-3,6-diyl)bis(ethene-2,1-diyl))bis(1-methylpyridin-1-ium)

(126) 3,6-bis((E)-2-(pyridin-4-yl)vinyl)-9-(4-((E)-2-(pyridin-4-yl)vinyl)phenyl)-9H-carbazole (176.5 mg, 0.32 mmol) was weighed into round bottom flask and dissolved in a DCM/MeOH (1:1 v:v) mixture. Methyl iodide (6 mL, 96.39 mmol) was then added. The solution was heated at reflux for 3 days and cooled to room temperature. The red precipitate (108.3 mg, 35%) was filtered and dried.

(127) .sup.1H NMR (DMSO, 400 MHz) 4.28 (s, 6H), 4.31 (s, 3H), 7.59-7.71 (m, 5H), 7.87 (d, 2H, J=8.0 Hz), 7.96 (d, 2H, J=2, 9.2 Hz), 8.11 (d, 1H, J=8.0 Hz), 8.17-8.21 (m, 2H), 8.25 (d, 4H, J=8.0 Hz), 8.28 (s, 1H), 8.30 (d, 2H, J=4.0 Hz), 8.69 (s, 1H), 8.76 (s; 1H), 8.86 (d, 5H, J=8.0 Hz), 8.93 (d, 2H, J=8.0 Hz). .sup.13C NMR (100 MHz, DMSO) 152.7, 145.3, 145.0, 141.7, 139.3, 135.0, 130.0, 128.6, 127.2, 124.4, 123.7, 123.5, 123.1, 121.4, 111.1, 47.0, 46.8.

Example 5a: Synthesis of Pyrene-Based Fluorescent Molecular Rotor

(128) ##STR00148##

(129) Pyrenecarboxaldehyde (0.5 g, 2.17 mmol, 1.0 eq.) and cyanoacetic acid (0.277 g, 3.26 mmol, 1.5 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.60 mL, 4.34 mmol, 2.0 eq.) was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 55 C. overnight, then evaporated to dryness and purified via column chromatography to yield pale orange solids (0.2 g, 31%).

(130) .sup.1H NMR (DMSO, 400 MHz) 8.14 (t, J=7.6 Hz, 1H), 8.23 (d, 1H, J=8.9 Hz), 8.30 (d, 1H, J=8.7 Hz), 8.34 (d, 1H, J 7.5 Hz), 8.39 (m, 3H), 8.57 (d, 1H, J=8.1 Hz), 9.00 (s, 1H). .sup.13C NMR (100 MHz, DMSO) 162.8, 145.8, 132.2, 130.7, 130.1, 129.1, 128.8, 127.9, 127.1, 126.7, 126.3, 126.1, 125.8, 124.9, 123.8, 123.6, 122.6, 120.4, 118.9.

Example 5b: Synthesis of Pyrene Rotor-Biotin

(131) ##STR00149##

(132) Preparation of Pyrene Biotin.

(133) Pyrenecarboxaldehyde (21.7 mg, 0.094 mmol, 1.0 eq.) and Biotin-CN (0.05 g, 0.141 mmol, 1.5 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.065 mL, 0.47 mmol, 5 eq.) was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 55 C. overnight, then evaporated to dryness and purified via column chromatography to yield pale orange solids (29.2 mg, 55%).

(134) .sup.1H NMR (MeOD, 400 MHz) 0.85 (m, 2H), 1.24 (s, 4H), 2.00 (m, 4H), 2.55 (m, 2H), 3.05 (m, 2H), 5.33 (t, 1H, J=4.8 Hz), 8.14 (t, J=7.6 Hz, 1H), 8.23 (d, 1H, J=8.9 Hz), 8.30 (d, 1H, J=8.7 Hz), 8.34 (d, 1H, J=7.5 Hz), 8.39 (m, 3H), 8.56 (d, 1H, J=8.3 Hz), 8.95 (s, 1H). HRMS (TOF MS ES+): calcd for C.sub.32H.sub.30KN.sub.4O.sub.4S [M+K].sup.+ 605.1988. found 604.9648.

Example 6a: Synthesis of Julodine-Based Fluorescent Molecular Rotor (CCVJ)

(135) ##STR00150##

(136) Julolidine-carboxaldehyde (0.5 g, 2.48 mmol, 1.0 eq.) and cyanoacetic acid (0.3170 g, 3.73 mmol, 1.5 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.69 mL, 4.97 mmol, 2.0 eq.) was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 55 C. overnight, then evaporated to dryness and purified via column chromatography to reddish-brown solids (0.18 g, 27%).

(137) .sup.1H NMR (DMSO, 400 MHz) 1.87 (m, 3H), 2.67 (t, J=6.3 Hz, 1H), 3.31 (t, J=5.9 Hz, 1H), 7.48 (s, 2H), 7.84 (s, 2H). .sup.13C NMR (100 MHz, DMSO) 165.1, 152.8, 147.1, 130.7, 120.4, 119.5, 118.5, 117.5, 104.5, 49.4, 27.0, 20.6.

Example 6b: Synthesis of Julodine Rotor-Biotin

(138) ##STR00151##

(139) Julolidine-carboxaldehyde (0.05 g, 0.25 mmol, 1.0 eq.) and Biotin-CN (0.1 g, 0.28 mmol, 1.13 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.07 mL, 0.5 mmol, 2.0 eq.) was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 50 C. overnight, then evaporated to dryness and purified via column chromatography to yield yellowish orange solids (27.8 mg, 18%).

(140) .sup.1H NMR (CDCl.sub.3, 400 MHz) 1.42 (m, 2H), 1.59 (m, 4H), 1.98 (m, 2H), 2.25 (m, 1H), 2.31 (m, 1H), 2.75 (t, 2H, J=6.3 Hz), 2.97 (dd, 1H, J=13.8, 5.4 Hz), 3.19 (m, 1H), 3.36 (m, 4H), 3.56 (m, 1H), 3.68 (m, 1H), 4.13 (d, 1H, J=8.0 Hz), 4.22 (t, 1H, J=5.9 Hz), 4.27 (m, 1H), 4.35 (dd, 2H, J=5.7, 4.3 Hz), 5.98 (s, 1H), 6.78 (s, 1H), 7.52 (m, 2H), 7.70 (dd, 1H, J=5.7, 3.3 Hz), 7.94 (s, 1H). .sup.13C NMR (100 MHz, CDCl.sub.3) 173.6, 165.1, 162.1, 155.0, 148.4, 132.0, 121.1, 118.3, 114.0, 90.0, 64.4, 62.2, 57.5, 54.9, 50.4, 39.1, 37.9, 36.6, 34.0, 27.7, 27.2, 25.4, 25.1, 21.1. HRMS (TOF MS ES+): calcd for C.sub.28H.sub.35N.sub.5NaO.sub.4S [M+Na].sup.+ 560.2302. found 560.2321.

Example 7: Fluorescent Molecular Rotors Bound to Ligands

(141) Molecular rotors can be modified to incorporate a targeting molecule or short targeting peptides. For example, biotin was attached to the molecular rotors and an increase in fluorescence was observed upon binding to the streptavidin protein.

(142) Molecular rotors may be conjugated to targeting peptides via a 3-carbon linker. Amide linkage may be used to conjugate the rotors via a lysine residue at the carboxy-terminus of targeting peptides.

Example 8: Viscosity Experiments with Fluorescent Molecular Rotors

(143) FIGS. 3a to 10 illustrate the emission spectras of fluorescent molecular rotors. It is shown that these fluorescent molecular rotors display an increase in fluorescence upon increase in viscosity. For all the rotors synthesized (julodine-based, aciridine-based, pyrene-based, thiazole-based and carbozole-based (2-arm and 3-arm)), the viscosity tests showed good sensitivity and trend.

Example 9: Binding of Rotor-Small Molecule with Protein

(144) A fluorescent julodine-based molecular rotor, 9-(2-carboxy-2-cyanovinyl) julolidine (CCVJ) was conjugated to biotin and changes in fluorescence was measured upon binding to streptavidin. A 2-fold fluorescence increase was measured upon the addition of streptavidin, demonstrating the utility of the biotin-rotor probe (FIG. 4a).

(145) A pyrene-based molecular rotor was also tested and displayed fluorescence increase upon binding to streptavidin (FIG. 4b).

Example 10: Binding of Rotor-DNA with Protein

(146) DNA Intercalation of Molecular Rotors

(147) Intercalation of DNA with fluorescent molecular rotors displayed a strong increase in fluorescence signal. The intercalating interaction with DNA was further exemplified through a pull-down assay with biotin-conjugated rotors and streptavidin beads (FIGS. 6, 7 and 16).

(148) FIGS. 4a and 4b illustrate that julodine-based and pyrene-based molecular rotors display an increase in fluorescence upon binding to Streptavidin protein demonstrating the usefulness of fluorescent molecular rotors as a probe for streptavidin protein.

(149) Molecular Rotors in Detection of p53-DNA Binding

(150) An acridine-based rotor was used in a fluorescence displacement assay to report on the binding of p53 protein to DNA. A decreased fluorescence signal was observed upon protein binding (FIG. 5).

(151) Protein-DNA Interactions

(152) The intercalating activity of fluorescent molecular rotors was tested on the binding sequence of p53 protein. It was shown that the fluorescence intensity of the rotors increase steadily until they reached saturation at 0.5 rotor for acridine-based rotor, and 15 rotor for thiazole-based and carbazole-based (2-arm and 3-arm) rotors (FIG. 7).

(153) From the trend given by the emission spectras of rotor with DNA, it can be concluded that the rotors do intercalate or bind to the DNA shown by the increase in fluorescence intensity due to the restriction of rotor motion upon intercalating or binding.

(154) The use of rotors in protein-DNA interactions was then tested.

(155) When a significant portion of p53 protein was added to acridine-based rotor bound to DNA, the fluorescence intensity dropped. It is hypothesized that upon interaction and binding of p53 with the DNA, the bulky acridine-based rotor intercalator was freed from the intercalating site, resulting in the reduction of fluorescence intensity (FIG. 8).

(156) p53 protein was added to carbazole-based (2-arm) rotor bound to DNA. A turn-off effect was observed when p53 was added, with effect most significant when the DNA was saturated with the 15 compound (FIG. 10).

(157) The behavior of rotors when incubated with DNA was also examined (FIG. 12).

(158) Whether the binding of a protein (p53) to DNA (with rotor bound) resulting in any fluorescence change due to displacement of the rotor by the protein was also examined. P53 protein was added to either control DNA (not containing a p53 binding motif) or DNA containing the p21 response element which p53 is known to bind. The data indicates that p53 binding to p21 can result in displacement of the rotor with concomitant reduction in fluorescence (FIG. 15).

(159) Whether biotin modification would affect binding to DNA was examined. It was also examined whether the addition of biotin would make rotors useful for affinity purification of DNA. Biotinylated versions of the rotors were incubated with DNA and complexes captured on streptavidin plates. Captured DNA was then quantified by real-time PCR. The results in FIG. 16 indicate that these rotors can be used as reagent for affinity purification of DNA. The fluorescent properties of the biotinylated rotor changed upon binding to streptavidin (FIG. 6), presumably due to steric constraints imposed upon binding of the biotin.

Example 11: Binding of Rotor-Peptide with Protein

(160) Synthesis of Rotor-Peptide Probes, JP1-R and JP2-R

(161) A julodine-based rotor, CCVJ, was conjugated to two peptides derived from a phage display screen, JP1 and JP2, which differ by only a single amino acid but have a fold difference in binding affinities (Table 9). An additional lysine residue was added to the C-termini of both peptide sequences, then reacted with the N-hydroxysuccinimidyl-activated ester of CCVJ to make the rotor-peptide probes (FIG. 25a).

(162) Unmodified peptides JP1 and JP2 were synthesized by Genscript USA Inc. Chemicals and solvents were purchased from Sigma Aldrich and TCI Chemicals Japan.

(163) JP1 (2 mg) was weighed into a 1.5 mL vial and dissolved in 0.3 mL of 0.1M NaHCO.sub.3 buffer. NHS-protected CCVJ (4 equiv.) was also weighed into another 1.5 mL vial and dissolved in 0.1 mL dry DMF, before being added to the JP1 that was dissolved in buffer. The reaction mixture was allowed to stir at room temperature for 2 hours before HPLC purification to obtain the pure rotor-peptide, JP1-R (9%). MALDI-TOF MS [M]+ Calculated 1949.8957. Obtained 1946.6082.

(164) JP2 (2 mg) was weighed into a 1.5 mL vial and dissolved in 0.3 mL of 0.1M NaHCO.sub.3 buffer. NHS-protected CCVJ (4 equiv.) was also weighed into another 1.5 mL vial and dissolved in 0.1 mL dry DMF, before being added to the JP2 that was dissolved in buffer. The reaction mixture was allowed to stir at room temperature for 2 hours before HPLC purification to obtain the pure rotor-peptide, JP2-R (4%). MALDI-TOF MS [M+K].sup.+ Calculated 2015.8703. Obtained 2017.6360.

(165) JP2 (2 mg) was weighed into a 1.5 mL vial and dissolved in 0.3 mL of 0.1M NaHCO.sub.3 buffer. NHS-protected CCVJ rotor (4 equiv.) was also weighed into another 1.5 mL vial and dissolved in 0.1 mL dry DMF, before being added to the JP2 that was dissolved in buffer. The reaction mixture was allowed to stir at room temperature for 2 hours before HPLC purification to obtain the pure rotor-peptide, JP2-R (4%). MALDI-TOF MS [M+K].sup.+ Calculated 2015.8703. Obtained 2017.6360.

(166) Rotor-peptide probes, JP1-R and JP2-R, are shown in FIGS. 25b and 25c.

(167) Binding of Rotor-Peptide Conjugates to a Protein

(168) Affinity purified recombinant MDM2 protein (residues 18-125) was used to test the functionality of the rotor-peptide conjugates. Co-incubation of JP1-rotor conjugate, JP1-R, with MDM2 protein, led to a concentration dependent increase in fluorescence activity (FIG. 20a (top)).

(169) Nutlin-3, an MDM2 agonist, binds MDM2 at the N-terminal hydrophobic cleft and abrogates this interaction by occluding p53-MDM2. Addition of 50 uM of Nutlin to the JP1-R-MDM2 complex MDM2 completely abrogated the florescence signal seen before (FIG. 20a, Bottom), presumably due to the displacement of JP1 from MDM2. JP2-R did not display any significant changes in fluorescence intensity upon adding MDM2. This lack of signal May be ascribed to the non-constrained orientation of the rotor upon binding of the peptide to MDM2.

(170) To further demonstrate that fluorescence activation was due to a concomitant steric restriction of the appended rotor during protein-specific interaction, JP1-R was added to non-specific proteins, eIF4E, BSA and IgG. No fluorescence increase was observed with all 3 proteins across the same concentrations range (FIG. 20b).

(171) Based on the JP1-R fluorescence measurements, an apparent K.sub.d of 16.017.52 nM for MDM2 binding was calculated (FIG. 21a), correlating well to a previously reported value of 18.835.03 nM (Table 9) determined using ITC. The calculated apparent K.sub.d of JP2-R, was 3365640.6 nM, approximately 14-fold lower than the previously reported value of 239.8153.79 nM.

(172) To further understand the fluorescence-derived apparent dissociation constants of JP1-R and JP2-R, an in-silico modeling of their respective interactions with the MDM2 protein was performed. Molecular dynamics simulations suggest that the C-terminal end of JP1-R adopts a helical turn due to the constraints from the hydrogen bonds between the hydroxyl sidechain and backbone of S12 and the backbone carbonyl of W8. A similar feature was also seen in prior experimental and computational studies for a similar peptide. The replacement of S12 by N12 in JP2-R does not afford this constraint. The Asn sidechain is longer and is unable to form hydrogen bonds with the backbone, resulting in an extended C-terminus (FIG. 27). Replica exchange simulations exploring the conformational space of the unbound rotor-peptides (Table 4) show that JP1-R is more helical than JP2-R. The constrained JP1-R (FIG. 27) also embeds deeper into MDM2 and interacts stronger than JP2-R (by 7kT, Table 5). The major contribution arises from improved packing of S12 (by 1.2kT, Tables 6, 7), L11 (embeds deeper into MDM2 by 2kT) and the rotor (2kT). The rotor packs between H96 of MDM2 and the peptide in JP1-R while JP2-R packs into a recently characterized second binding site in MDM2. This tighter association of JP1-R restricts the rotational freedom of the rotor sufficiently to bring about a detectable fluorescence turn-on signal.

Example 12: Drug Screening Applications

(173) Rotor-peptide may have potential use in drug-screening applications. Small molecule inhibitor nutlin was titrated and a disruption of the rotor-peptide-MDM2 complex at concentrations as low as 10 nM was observed (FIG. 11). The results indicate highly sensitive detection of nutlin binding to MDM2, suggesting the rotor-peptide assay may have use in primary drug screens for disruption of a peptide-protein interaction.

(174) Rotor-Peptide Conjugate as a Biosensor for Small Molecules that Inhibit p53-MDM2 Interaction

(175) A concentration-dependent drop in fluorescence was seen when nutlin was titred into a pre-incubated mix containing 500 nM of MDM2 and 250 nM of JP1 (FIG. 11). Complete ablation of JP1-MDM2 dependent fluorescence was observed at 2 uM of Nutlin treatment, but a significant decrease can be seen at 50 nM. The sensitivity and specificity of such peptide-rotor conjugates make them ideal for development into screening platforms for identifying molecules that inhibit p53-MDM2 binding.

(176) Application of Rotor-Peptide in Small Molecule Drug Screening (E.G. Inhibitors of MDM2)

(177) The inhibition of p53-MDM2 interaction is a common strategy for cancer treatment. A rotor is attached to the JP1 peptide (a sequence that binds the MDM2 protein at the same site as p53) to investigate if the JP1 peptide-rotor conjugate will act as a reporter for compounds that displace it from MDM2. FIG. 23 shows that such a displacement can be seen when various known p53-MDM2 inhibitors are added to the JP1/MDM2 complex. Calculated K.sub.d from MDM2 against JP1 titration indicates that rotor-moeity does not obstruct peptide-protein interaction (FIG. 21a).

(178) To demonstrate the utility of the rotor-peptide probe in small molecule drug screening, the sensitivity of the rotor-peptide to known small molecule and stapled-peptide inhibitors of the p53-MDM2 interaction was explored. These inhibitors target the same hydrophobic cleft in MDM2 as JP1-R, and were able to disrupt the MDM2-probe complex in the expected manner, resulting in a decrease in fluorescence as the probe was displaced (FIG. 24 and Table 8).

(179) Given the high sensitivity and specificity of the JP1-R conjugate, it was used to screen a small molecule fragment library (n=352) for candidates that potentially disrupt p53-MDM2 binding. Based on the results, fifteen hits were selected for further validation and ten compounds were further confirmed as genuine inhibitors (FIG. 26b). The seven disparate compounds showing no activity in the FP assay were therefore assessed in a pull-down assay which measured the direct interaction levels of p53 and MDM2. All seven compounds inhibited p53-MDM2 interaction, further validating these as genuine hits not identified by the FP assay (FIG. 26c). As subtle intermolecular twisting of the rotor profoundly effects signal generation, it is possible that partial displacement of the peptide by the weak inhibitors led to their identification. In the FP assay, these would have been missed, as the anisotropy measurement is largely attendant on full displacement of peptide from MDM2.

(180) Together, these results demonstrate the utility of molecular rotors in binding assays for detecting peptide-protein interactions and for drug screening applications. Using the TICT property of the molecular rotor, its free volume is decreased upon binding interaction to a protein. This simple fluorescence turn-on signal upon protein binding allows the development of highly sensitive and facile assays to measure protein-ligand binding in a high-throughput fashion. More importantly, it is shown that a molecular rotor-based screening assay identified validated hits that were missed by fluorescence polarization assay in a fragment-based screen, suggesting its utility in identifying lower affinity hits in fragment based screening.

APPLICATIONS

(181) The disclosed fluorescent molecular rotors may be used to detect protein-ligand interactions, such as protein-DNA, protein-peptide and protein-small molecule interactions, for example, to investigate the interaction of p53 protein with DNA, interaction of MDM2 with peptides. The disclosed fluorescent molecular rotors may also be conjugated to biotin to give a streptavidin detection reagent.

(182) The disclosed fluorescent molecular rotors may be used as viscosity probes, displaying an increase in fluorescence when viscosity of the solution is increased.

(183) The disclosed fluorescent molecular rotors may be used in drug screening applications. The disclosed fluorescent molecular rotors may be used to screen for active compounds that bind to a target protein, for example, inhibitors of MDM2.

(184) The disclosed screening applications may not require the use of expensive instrumentation. Thus, the disclosed screening applications may serve as a cost-effective means for drug screening.

(185) The disclosed screening applications may be single-well, low volume with minimal pipetting steps and may not require multiple washing steps. Thus, the disclosed screening applications may be non-laborious and optimal for high-throughput screening.

(186) The disclosed screening applications may not require the use of radioisotopes for labelling. Thus, the disclosed screening applications may be non-radioactive.

(187) The disclosed screening applications may be used in small molecule drug screening and may be used to identify small molecules which may be missed in traditional assays, such as fluorescence polarization.

(188) It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.