The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue

Figures & data

Figure 1. (A) Structures of PYO and MB in oxidized and reduced forms. (B) PYO and MB as redox cycling catalysts in vivo: NAD(P)H-dependent reduction of the blue oxidized forms and rapid re-oxidation of the leuco forms by auto-oxidation. (C) PYO and MB as diaphorase substrates of P. falciparum mitochondrial lipoamide dehydrogenase. In the Lineweaver–Burk diagram, the ordinate axis intercepts represent the reciprocal values of the specific activity for PYO (squares) and MB (circles). The enzyme preparation described in the experimental section was used. The OH group of PYO_ox has a pK_a of 4.9.

Figure 2. Spectra of PYO in oxidized and NADPH-reduced form in 50 mM potassium phosphate buffer of pH 7. The peak at 340 nm in the reduced spectrum (dotted line) is largely due to excess NADPH.

Table 1. Properties of PYO in comparison to MB

Property	Pyocyanin	MB
Chemical class	Phenazine	Phenothiazine
Color	Blue	Dark blue
Colorless reduction product	LeucoPYO	LeucoMB (solubility only 25 µM)
λvis,max	690 ± 100 nm	663 nm
Mr of the heterocycle	210 Da	284 Da
Milestones	Forbes 1860, Wrede 1924, 1929, Friedman and Michaels 1931, Newman 2006	Caro 1884, Bernthsen 1887, Ehrlich 1891, Wieland 1922, Clark 1925
Functions in vivo	In P. aeruginosa respiratory pigment, quorum sensor, transcription factor, antimicrobial agent^Citation1	Numerous technical, industrial, scientific and medical applications
Clinical dosage for various diseases	Not applicable	3 mg per kg per day^Citation22
Concentrations in vivo	<100 µM in infected bronchial mucus (see Ref 5)	5–30 µM in whole blood^Citation22
Midpoint potential at pH 7	−34 mV^Citation24 (see Acknowledgements)	+10 mV
Intracellular reductants (k values in M^−1s^−1 at pH 7 and 25°C)	NADPH (46) > NADH (26) >dihydrolipoamide (3.2) >GSH (<0.5)	Dihydrolipoamide (53) > thioredoxin (27) > NADPH (6.6) >NADH (3.5) >GSH (<0.5)^Citation19
Redox cycling catalyst in vivo	Yes, reduction by NADPH, reoxidation by O2	Enzyme-catalyzed reduction by NADPH, reoxidation by O2
Reduction by flavoenzymes	LipDH and TrxR	LipDH >TrxR > GR
Inhibition of flavoenzymes	GR and TrxR, not LipDH	GR and TrxR, not LipDH
Crystal structure of enzyme-ligand complex	Human GR-PYO complex	Low resolution data^Citation25
IC50 against P. falciparum blood stages in vitro	86 ± 4.5 nM [3D7 strain]	3.1 ± 0.4 nM [3D7 strain]
	59 ± 3.3 nM [K1 strain]	6.6 ± 1.1 nM [K1]
IC50 against early (and mature) P. falciparum gametocytes in vitro	180 nM	34 nM
	(560 nM)	(60 nM)
Tested for biological conduct in vivo	In >100 Ma of evolution in bacteria-containing biotopes	In an unprecedented variety of uses in medicine and biotechnology

Data on PYO listed from line ‘Redox-cycling catalyst’ to the bottom line are from this paper. This applies also to the IC₅₀ values of MB; most other data on MB were taken from Ref 19. IC₅₀ is the concentration of an inhibitor where biological and/or biochemical activity is reduced to 50%.

Table 2. Enzyme-kinetic data for interactions of PYO and MB with disulfide reductases

Enzyme	KM for PYO (μM)	kcat for PYO (second^−1)	kcat/KM (M^−1 second ^−1)	IC50 for PYO (μM)	KM of MB (μM)	kcat for MB (second ^−1)	kcat/KM (M^−1 second ^−1)	IC50 for MB (μM)
Human GR	43	0.17	3950	7	6.3	0.03	4760	16.4
Human GR C63A	600	3.9	6500	n.a.	56	0.62	11 100	n.a.
Pf GR	38	0.03	790	15	50	2.50	50 000	5.4
Porcine LipDH	25	1.2	48 000	>500	28	3.20	114 000	>1000
Pf mtLipDH	60	0.9	15 000	>500	31	4.00	129 000	>1000
Human TrxR	170	>0.9	>5300	n.d.	95	10.00	105 000	30
Pf TrxR	31	0.7	22 600	7	68	6.00	88 200	59

P. falciparum GR has a catalytic efficiency of only 790 M⁻¹ second⁻¹ at pH 8.0 and even lower values at physiological pH values. The abbreviations are Pf, P. falciparum; GR, GR; LipDH, dihydrolipoamide dehydrogenase; TrxR, thioredoxin reductase; n.a., not applicable, because the C63A mutant of GR, lacking one catalytic-site cysteine, has no disulfide reductase activity. Most data for MB are from.¹⁹ Not listed is the enzyme-catalyzed auto-oxidation rate which refers to the reaction between NAD(P)H and atmospheric O₂. It ranges from 0.003 second⁻¹ for human GR to 0.260 second⁻¹ for P. falciparum mitochondrial LipDH.

Figure 3. Human GR homodimer with bound PYO. PYO (blue) and FAD (yellow) are represented as ball-and-stick models. Additionally, the surfaces of the catalytic cysteines Cys58/Cys63 and Cys58′/Cys63′ (yellow) and of PYO (blue) are shown. The atomic coordinates and their structure factors determined by x-ray diffraction analysis have been deposited at the PDB (Protein Data Bank) under the accession code (PBD ID) 3SQP. Molecular graphics images were produced using the UCSF Chimera package.²⁶

Figure 4. Binding of PYO to human GR. For clarity only one of the two observed orientations of PYO (blue carbon atoms) is shown. Green C_α-atoms indicate amino acids at distances of less than 5 Å from the ligand. O atoms are shown in red, N atoms in blue, and sulfur atoms in yellow.

Table 3. Summary of data collection and structure refinement of the crystalline GR–PYO complex

Protein	Human GR
Space group	P1
Unit cell dimensions (Å)	a = 62.08, b = 67.37, c = 78.86
	α = 68.98°, β = 66.8°, γ = 52.54°
Resolution range (Å)*	37.71–2.21 (2.27–2.21)
Completeness (%)	93.9 (82.4)
Mean redundancy	3.3 (3.0)
Rsym** (%)	2.9 (6.2)
(I/σ)	29.0 (17.4)
Reflections used in refinement	47 819
Protein atoms	7116
Solvent molecules	858
rmsd bonds (Å)/rmsd angles	0.01/0.9
Rcryst*** (%)/Rfree**** (%)	14.3/18.1

*Values within parentheses represent data in the last resolution shell.

**R_sym = 100∑|I_h–|/∑I_h.

***R_cryst = 100∑‖F_obs|–|F_calc‖/∑|F_obs| calculated for all observed data. No sigma cut-off was used.

****R_free calculated for 3% of randomly chosen unique reflections that were excluded from the refinement.

Figure 5. Stage-specific effects of various concentrations of PYO (x-axis, given in nM) on different developmental stages of the asexual forms of P. falciparum in cultured erythrocytes. The data indicate a rapid antiparasitic effect, with the schizont stage exhibiting the highest susceptibility.

Table 4. In vitro activities of PYO, MB, and CQ against immature (day 9) and mature (day 13) P. falciparum gametocytes

Compound	IC50 (95% CI) [nM]
	Day 9 gametocytes (stages II and III)	Day 13 gametocytes (stages IV and V)
PYO	178 (147–215)*	557 (262–1190)
MB	33.8 (32.1–35.7)	59.5 (37.3–94.8)
CQ	8.60 (3.02–24.7)	487 (278–854)

*95% Confidence interval. The IC₅₀s between early (day 9, stages II and III) and late (day 13, stages IV and V) gametocyte stages were significantly different for all compounds, the P-values being 0.0021 for PYO, 0.0013 for MB, and 0.0146 for CQ.

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