N-(3-hydroxymethyl-β-carboline-1-yl-ethyl- 2-yl)-l-Phe: development toward a nanoscaled antitumor drug capable of treating complicated thrombosis and inflammation

Figures & data

Figure 1 Structural similarity of N-[2(3-carboxyl-9-benzyl/H-carboline-1-yl)ethyl-1-yl]-amino acids, BPIC, PZL318, NRCB and HMCEF.

Abbreviations: Arg, argnine; BPIC, benzyl 1-(4-hydroxy-3-methoxycarbonyl-phenyl)-9H-pyrido[3,4-b]indole-3-carboxylate; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; Lys, lysine; NRCB, benzyl N^ω-nitro-N^α-(9H-pyrido[3,4-b]indole-3-carbonyl)-L-argininate; OBzl, benzyl ester; PZL318, N-[1-(3-methoxycarbonyl- 4-hydroxyphenyl)-β-carboline-3-carbonyl]-Trp-Lys-OBzl; Trp, tryptophan.

Figure 2 Docking feature of HMCEF in the active site of d(CGATCG)₂.

Abbreviation: HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe.

Figure 3 Absorbance changes after interaction of HMCEF and CT DNA.

Abbreviations: CT DNA, calf thymus DNA; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe.

Figure 4 Fluorescence spectra of HMCEF.

Notes: (A) Fluorescence spectra of HMCEF in PBS buffer (concentration: 0.5 μM, pH =7.4, λ_ex =254 nm) explain the fluorescence quenching induced by 10 μL of CT DNA (final concentrations: 0, 8, 16, 24, 32, 40, 48, 56, 64 and 72 μM) in PBS. (B) Fluorescence spectrum of EB plus CT DNA and HMCEF (the concentration of EB is 5 μM, the concentration of DNA is 10 μM and the concentrations of HMCEF are 0, 10, 20, 30, 40, 50, 60 and 70 μM, respectively).
Abbreviations: CT DNA, calf thymus DNA; EB, ethidium bromide; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; PBS, phosphate-buffered saline.

Figure 5 CD spectra of CT DNA in the absence (curve a) and presence of HMCEF (curve b) at (HMCEF)/(CT DNA) value of 2.0.

Abbreviations: CD, circular dichroism; CT DNA, calf thymus DNA; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe.

Figure 6 Thermal denaturation curves of CT DNA with and without HMCEF.

Note: T_m measurements were performed in PBS at pH 7.4 with HMCEF/DNA ratio of 4.34; A₀ is the initial absorbance. A_f is the final absorbance, and A is the absorbance at any temperature.

Abbreviations: ΔT_m, shift of the melting temperature; CT DNA, calf thymus DNA; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; PBS, phosphate-buffered saline.

Figure 7 Effect of increasing amounts of HMCEF on the relative viscosity of CT DNA at 30°C.

Notes: η is the viscosity of DNA in the presence of HMCEF; η₀ is the absence of HMCEF; C_HMCEF is the concentration of HMCEF; and C_{CT DNA} is the concentration of CT DNA.

Abbreviations: CT DNA, calf thymus DNA; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe.

Figure 8 Electrophoretogram of supercoiled DNA plasmid pBR322 and HMCEF treated plasmid pBR322 DNA.

Notes: Lane 1, electrophoresis strip of plasmid pBR322 DNA without HMCEF. Lane 2, electrophoresis strip of supercoiled plasmid pBR322 DNA plus 100 μM of HMCEF. Lane 3, electrophoresis strip of supercoiled plasmid pBR322 DNA plus 200 μM of HMCEF. Lane 4, electrophoresis strip of supercoiled plasmid pBR322 DNA plus 300 μM of HMCEF. Lane 5, electrophoresis strip of supercoiled plasmid pBR322 DNA plus 400 μM of HMCEF. Lane 6, electrophoresis strip of supercoiled plasmid pBR322 DNA plus 500 μM of HMCEF. Form I, supercoiled DNA. Form II, nicked DNA.
Abbreviation: HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe.

Figure 9 FT-MS, qCID and ROESY 2D NMR spectra, as well as the conformation of HMCEF.

Notes: (A) FT-MS spectrum and the insert of HMCEF give an ion peak of monomer plus H at 390.18013, an ion peak of tetramer plus H at 1,557.69833 and a divalent ion peak of octamer plus H at 1,557.69833. The inset shows magnified area identified by the red square. (B) qCID spectrum of the octamer gives ion peaks of the monomer plus H, the dimer plus H, the trimer plus H, the tetramer plus H and the pentamer plus H. (C) Local amplified spectrum gives an ion peak of the pentamer plus H at 1,946.88254. (D) ROESY 2D NM Rspectrum and two interesting cross-peaks labeled with red circles and named 1 and 2. (E) Energy-minimized conformation of the monomer. (F) Energy-minimized bougarabou-like conformation of the octamer.
Abbreviations: FT-MS, Fourier transform mass spectrometry; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; Intens, intensity; NMR, nuclear magnetic resonance; qCID, qualitative contingent influence diagram; ROESY, rotating-frame Overhauser effect spectroscopy.

Figure 10 TEM, SEM and AFM images of HMCEF, as well as the prediction of a nanoparticle of 10.7 nm in diameter by mesoscale simulation.

Notes: (A) TEM image of HMCEF in ultrapure water (pH 7.0, 10⁻⁶ M) and amplified nanoparticle of 10 nm in diameter, which has a porous surface. (B) SEM image of the lyophilized powders from 10⁻⁶ M solution of HMCEF in ultrapure water (pH 7.0, 10⁻⁶ M) and amplified nanoparticle, which has a porous surface. (C) AFM image of rat plasma alone, and gives no comparable particle. (D) AFM image of HMCEF in ultrapure water (10⁻⁶ M). D1 scales the particle inside the blue ring; D2 scales the particle inside the yellow ring, and the magnified area identified by the red triangles. Red triangles are plot markers; black lines are grid cursors. (E) AFM image of HMCEF in rat plasma (10⁻⁶ M). E1 scales the particle inside the pink ring, and the magnified area identified by the red triangles. In D and E the red triangles are plot markers and the black lines are grid cursors. (F) Magnification of the particle inside the blue ring of D to show its morphology. (G) Magnification of the particle inside the yellow ring of D to show its morphology. (H) Magnification of the particle inside the pink ring of E to show its morphology. (I) Mesoscale simulation predicts that a nanoparticle of HMCEF contains 206 octamers.
Abbreviations: AFM, atomic force microscopy; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 11 HMCEF inhibits A549 cell-induced platelet aggregation in vitro.

Abbreviations: HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; NS, normal saline.

Figure 12 IC₅₀ of HMCEF against Bel7402, HeLa, HepG₂ and K562 cells and HMCEF dose-dependent inhibition of tumor growth in S180 mice.

Notes: (A) IC₅₀ of HMCEF against Bel7402, HeLa, HepG₂ and K562 cells; data are represented as mean ± SD (μM), n=6. (B) HMCEF dose dependently slowing tumor growth of S180 mice, n=12.
Abbreviations: HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; IC₅₀, half maximal inhibitory concentration.

Figure 13 Serum ALT, AST and Cr of S180 mice treated with 200 nmol/kg per day of HMCEF or NS for 10 consecutive days, n=12.

Notes: (A) Serum ALT of S180 mice treated with HMCEF or NS. (B) Serum AST of S180 mice treated with HMCEF or NS. (C) Serum Cr of S180 mice treated with HMCEF or NS; n=12.
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; Cr, creatinine; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; HMCEF, N-(3-hydroxymethyl-βcarboline-1-yl-ethyl-2-yl)-l-Phe; NS, normal saline.

Figure 14 Serum ALT, AST and Cr of healthy ICR mice treated with 200 nmol/kg of HMCEF or NS, n=12.

Notes: (A) Serum ALT of healthy ICR mice treated with HMCEF or NS. (B) Serum AST of healthy ICR mice treated with HMCEF or NS. (C) Serum Cr of healthy ICR mice treated with HMCEF or NS; n=12.
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; Cr, creatinine; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; HMCEF, N-(3-hydroxymethyl-β-carboline-1-yl-ethyl-2-yl)-l-Phe; NS, normal saline.