Cho Zin Soe

Zn(II)-dependent histone deacetylase inhibitors: Suberoylanilide hydroxamic acid and trichostatin A

Suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) and trichostatin A (TSA) are inhibitors of the Zn(II)-dependent class I and class II histone deacetylases (HDACs), which are enzymes that operate in concert with histone acetyltransferases (HATs) to regulate the acetylation status of the epsilon-amino group of lysine residues of nucleosomal histones in chromatin. An increased level of histone acetylation resulting from the SAHA or TSA inhibition of Zn(II)-dependent HDACs relaxes the chromatin structure and upregulates transcription. The links made in the 1990s between the inhibition of HDAC activity and the suppression of tumor growth have brought the design of HDAC inhibitors (HDACi) to the forefront of oncology research. SAHA has anticancer activity against hematologic and solid tumors and has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The increased molecular-level understanding of class I and class IIa HDACs from X-ray crystallography highlights differences in the residues distal to the active site and in the cavity size, which has implications for HDACi substrate specificity and enzyme mechanism. Results from HDAC-focussed activity-based protein profiling experiments may lead to the design of molecules that are class-specific HDACi.

Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.

Unsaturated macrocyclic dihydroxamic acid siderophores produced by Shewanella putrefaciens using precursor-directed biosynthesis.

To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H(+)](+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; [M + H(+)](+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H(+)](+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H(+)](+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)](+); [M](+), m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 ([Fe((15)N4-pb)](+); [M](+), m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)](+); [M](+), m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 ([Fe(E-pbx)](+); [M](+), m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical conversions across the double bond(s) of the unsaturated macrocycles provides a new route to increased molecular diversity in this class of siderophore.

The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

The recently sequenced genome of the marine actinomycete Salinispora tropica CNB-440 revealed a high frequency of gene clusters which code for the biosynthesis of known and novel secondary metabolites. Of these metabolites, bioinformatics analysis predicted that S. tropica CNB-440 could potentially biosynthesize, as high affinity Fe(iii) ligands, siderophores from the hydroxamic acid desferrioxamine class (sid1 gene cluster) and the phenolate-thia(oxa)zoli(di)ne class (sid2 and sid4 gene clusters). In this work, we have used Ni(ii)-based immobilized metal ion affinity chromatography (IMAC) to pre-fractionate the hydroxamic acid siderophore metabolome of S. tropica CNB-440 from the secondary metabolome, to reveal low abundance siderophores. LC-MS measurements and electronic absorption spectra from purified extracts incubated with exogenous Fe(iii) revealed eight siderophores from the desferrioxamine class (DFOA2, DFOA1a, DFOA1b, DFOB, DFON, DFOD2, DFOE, DFOD1), which included two constitutional isomers (DFOA1a, DFOA1b), and one new siderophore (DFON), the latter which would require assembly from a combination of 1,5-diaminopentane and 1,6-diaminohexane as diamine substrates. Three additional species (m/zobs 496.14, 792.34 and 804.34) with electronic absorption spectra characteristic of complexes formed between Fe(iii) and hydroxamic acid-type siderophores were evident under some conditions. The signal at m/zobs 792.34 eluted in the hydrophobic region of the reverse-phase LC and correlated with a DFOD1 analogue with a C-terminal branched chain fatty acid ([M + K(+)](+)m/zcalc 792.35), which has been previously identified from marine sediment dwelling Micrococcus luteus KLE1011. The S. tropica CNB-440 hydroxamic acid siderophore metabolome was modulated by culture conditions (pH 7, 22 °C; pH 7, 28 °C; pH 9, 28 °C) designed to simulate the variable marine environment. An increase in temperature at constant pH value showed increased levels of DFOA2 and DFOA1, and decreased levels of DFOB and DFOE. An increase in pH value at constant temperature showed decreased levels of DFOA2 and DFOA1, and increased levels of DFOB, DFON and DFOE. These results indicate that the marine adaptation of S. tropica CNB-440 could involve its ability to select a suite of siderophores from a large number of candidates, which are optimized for the iron microenvironment.

Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V).

Cultures of Shewanella putrefaciens grown in medium containing 10mM 1,4-diamino-2-butanone (DBO) as an inhibitor of ornithine decarboxylase and 10mM 1,5-diaminopentane (cadaverine) showed the simultaneous biosynthesis of the macrocyclic dihydroxamic acids: putrebactin (pbH2), avaroferrin (avH2) and bisucaberin (bsH2). The level of DBO did not completely repress the production of endogenous 1,4-diaminobutane (putrescine) as the native diamine substrate of pbH2. The relative concentration of pbH2:avH2:bsH2 was 1:2:1, which correlated with the substrate selection of putrescine:cadaverine in a ratio of 1:1. The macrocycles were characterised using LC-MS as free ligands and as 1:1 complexes with Fe(III) of the form [Fe(pb)]+, [Fe(av)]+ or [Fe(bs)]+, with labile ancillary ligands in six-coordinate complexes displaced during ESI-MS acquisition; or with Mo(VI) of the form [Mo(O)2(pb)], [Mo(O)2(av)] or [Mo(O)2(bs)]. Chromium(V) complexes of the form [CrO(pb)]+ were detected from solutions of Cr(VI) and pbH2 in DMF using X-band EPR spectroscopy. Supplementation of S. putrefaciens medium with DBO and 1,3-diaminopropane, 1,6-diaminohexane or 1,4-diamino-2(Z)-butene (Z-DBE) resulted only in the biosynthesis of pbH2. The work has identified a native system for the simultaneous biosynthesis of a suite of three macrocyclic dihydroxamic acid siderophores and highlights both the utility of precursor-directed biosynthesis for expanding the structural diversity of siderophores, and the breadth of their coordination chemistry.

Directing the biosynthesis of putrebactin or desferrioxamine B in Shewanella putrefaciens through the upstream inhibition of ornithine decarboxylase.

To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine‐depleted cultures. Compared to base medium, PB increased by two‐fold in cultures of S. putrefaciens with 10 mM NaCl and 20 mM exogenous putrescine. In cultures augmented with 1,4‐diaminobutan‐2‐one (DAB), PB decreased with only 0.02‐fold PB detectable at 10 mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine‐depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine‐based desferrioxamine B (DFO‐B), as characterised by ESI‐MS of the FeIII‐loaded form (m/zobs 614.13; m/zcalc 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the FeIII‐loaded extract gave m/zobs 440.93, which, formulated as a 1 : 1 FeIII complex with a macrocyclic dihydroxamic acid, comprising one putrescine‐ and one cadaverine‐based precursor (m/zcalc 440.14). These results show that the production of native PB or non‐native DFO‐B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine‐dependent metabolites.

Complexes Formed in Solution Between Vanadium(IV)/(V) and the Cyclic Dihydroxamic Acid Putrebactin or Linear Suberodihydroxamic Acid

An aerobic solution prepared from V(IV) and the cyclic dihydroxamic acid putrebactin (pbH2) in 1:1 H2O/CH3OH at pH = 2 turned from blue to orange and gave a signal in the positive ion electrospray ionization mass spectrometry (ESI-MS) at m/zobs 437.0 attributed to the monooxoV(V) species [VVO(pb)]+ ([C16H26N4O7V]+, m/zcalc 437.3). A solution prepared as above gave a signal in the 51V NMR spectrum at δV = −443.3 ppm (VOCl3, δV = 0 ppm) and was electron paramagnetic resonance silent, consistent with the presence of [VVO(pb)]+. The formation of [VVO(pb)]+ was invariant of [V(IV)]:[pbH2] and of pH values over pH = 2–7. In contrast, an aerobic solution prepared from V(IV) and the linear dihydroxamic acid suberodihydroxamic acid (sbhaH4) in 1:1 H2O/CH3OH at pH values of 2, 5, or 7 gave multiple signals in the positive and negative ion ESI-MS, which were assigned to monomeric or dimeric V(V)– or V(IV)–sbhaH4 complexes or mixed-valence V(V)/(IV)–sbhaH4 complexes. The complexity of the V-sbhaH4 system has been attributed to dimerization (2[VVO(sbhaH2)]+ ↔ [(VVO)2(sbhaH2)2]2+), deprotonation ([VVO(sbhaH2)]+ – H+ ↔ [VVO(sbhaH)]0), and oxidation ([VIVO(sbhaH2)]0 –e– ↔ [VVO(sbhaH2)]+) phenomena and could be described as the sum of two pH-dependent vectors, the first comprising the deprotonation of hydroxamate (low pH) to hydroximate (high pH) and the second comprising the oxidation of V(IV) (low pH) to V(V) (high pH). Macrocyclic pbH2 was preorganized to form [VVO(pb)]+, which would provide an entropy-based increase in its thermodynamic stability compared to V(V)–sbhaH4 complexes. The half-wave potentials from solutions of [V(IV)]:[pbH2] (1:1) or [V(IV)]:[sbhaH4] (1:2) at pH = 2 were E1/2 −335 or −352 mV, respectively, which differed from the expected trend (E1/2 [VO(pb)]+/0 < VV/IV–sbhaH4). The complex solution speciation of the V(V)/(IV)–sbhaH4 system prevented the determination of half-wave potentials for single species. The characterization of [VVO(pb)]+ expands the small family of documented V–siderophore complexes relevant to understanding V transport and assimilation in the biosphere.

Studies of iron-uptake mechanisms in two bacterial species of the shewanella genus adapted to middle-range (Shewanella putrefaciens) or antarctic (Shewanella gelidimarina) temperatures.

Iron(III)‐uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe‐deficient continent on Earth, have not been extensively studied. The cold‐adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of FeIII‐deprived cultures. This is distinct from the putrebactin‐producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle‐range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell‐associated siderophores. In the RP‐HPLC trace of an FeIII‐loaded extract from the cell‐associated components of S. gelidimarina cultured in media with [FeIII] ca. 0 μM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [FeIII] ca. 5 μM, and is barely detectable in FeIII‐replete media ([FeIII] ca. 20 μM). The FeIII‐dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP‐HPLC elution: [MeCN] ca. 14%), is associated with FeIII‐management in S. gelidimarina. This study highlights the diversity in FeIII‐uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.

Dinuclear [(VVO(putrebactin))2(μ-OCH3)2] Formed in Solution as Established from LC-MS Measurements Using 50V-Enriched V2O5

Analysis of 1:1 solutions of V(V) and the macrocyclic dihydroxamic acid siderophore putrebactin (pbH2) in 1:1 H2O/CH3OH using triple quadrupole liquid chromatography-mass spectrometry (LC-MS-QQQ) (pH ≈ 4) showed two well-resolved peaks (tR(1) 10.85 min; tR(2) 14.27 min) using simultaneous detection modes (absorbance, 450 nm; selective ion monitoring, m/z 437) characteristic of the previously identified oxidoV(V) complex [V(V)O(pb)](+) ([M](+), m/zcalc 437.1). Peak 1 gave mass spectrometry (MS) signals consistent with [V(V)O(pb)](+), together with [V(V)O(pb)(OH)] and the dinuclear complexes [(V(V)O(pb))2(μ-OH)](+) and [(V(V)O(pb))2(μ-OH)2]. Peak 2 gave MS signals consistent with [V(V)O(pb)](+), together with [V(V)O(pb)(OCH3)] and the dinuclear complexes [(V(V)O(pb))2(μ-OCH3)](+) and [(V(V)O(pb))2(μ-OCH3)2]. This analysis showed that two groups of V(V)/pbH2 complexes with water- or methanol-derived ancillary ligands were resolved by liquid chromatography (LC). The detection of [V(V)O(pb)](+) in both peaks could be accounted for by its production from dissociation (peak 1: [(V(V)O(pb))2(μ-OH)](+) → [V(V)O(pb)](+) + [V(V)O(pb)(OH)]; peak 2: [(V(V)O(pb))2(μ-OCH3)](+) → [V(V)O(pb)](+) + [V(V)O(pb)(OCH3)]). The assignment of the signal at m/zobs 959.2 (100%) as the dinuclear complex [(V(V)O(pb))2(μ-OCH3)2] ([M + Na(+)](+), m/zcalc 959.3) and not an ion cluster of mononuclear [V(V)O(pb)(OCH3)] ({2[M] + Na(+)}(+), m/zcalc 959.3) was made unequivocal by the use of (50)V-enriched V2O5, which gave a signal with an isotope pattern comprising the sum of the patterns of the three constituent (51)V-(51)V, (51)V-(50)V, and (50)V-(50)V species. Coordination of methoxide was confirmed upon the replacement of CH3OH with CD3OD, which generated [(V(V)O(pb))2(μ-OCD3)2] ([M + Na(+)](+), m/zcalc 965.3, m/zobs 965.3). Analysis of 1:1 solutions of Mo(VI) and pbH2 showed a single peak in the LC (tR 16.04 min), which gave MS signals that were characterized as mononuclear [Mo(VI)(O)2(pb)] ([M + Na(+)](+), m/zcalc 523.1, m/zobs 523.1) and dinuclear [(Mo(VI)O(pb))2(μ-O)2] ([M + Na(+)](+), m/zcalc 1019.1, m/zobs 1019.2). The steric and electronic effects of the cis-dioxido group(s) in [Mo(VI)(O)2(pb)] mitigated coordination of solvent-derived ancillary ligands. The work highlights the value of using isotopically enriched metal ion sources and deuterated solvents to deconvolute metal/siderophore solution speciation. The results have relevance for an improved understanding of the coordination chemistry of pbH2 and other marine siderophores in V(V)- and Mo(VI)-rich surface ocean waters.

The chemical biology and coordination chemistry of putrebactin, avaroferrin, bisucaberin, and alcaligin

Dihydroxamic acid macrocyclic siderophores comprise four members: putrebactin (putH2), avaroferrin (avaH2), bisucaberin (bisH2), and alcaligin (alcH2). This mini-review collates studies of the chemical biology and coordination chemistry of these macrocycles, with an emphasis on putH2. These Fe(III)-binding macrocycles are produced by selected bacteria to acquire insoluble Fe(III) from the local environment. The macrocycles are optimally pre-configured for Fe(III) binding, as established from the X-ray crystal structure of dinuclear [Fe2(alc)3] at neutral pH. The dimeric macrocycles are biosynthetic products of two endo-hydroxamic acid ligands flanked by one amine group and one carboxylic acid group, which are assembled from 1,4-diaminobutane and/or 1,5-diaminopentane as initial substrates. The biosynthesis of alcH2 includes an additional diamine C-hydroxylation step. Knowledge of putH2 biosynthesis supported the use of precursor-directed biosynthesis to generate unsaturated putH2 analogues by culturing Shewanella putrefaciens in medium supplemented with unsaturated diamine substrates. The X-ray crystal structures of putH2, avaH2 and alcH2 show differences in the relative orientations of the amide and hydroxamic acid functional groups that could prescribe differences in solvation and other biological properties. Functional differences have been borne out in biological studies. Although evolved for Fe(III) acquisition, solution coordination complexes have been characterised between putH2 and oxido-V(IV/V), Mo(VI), or Cr(V). Retrosynthetic analysis of 1:1 complexes of [Fe(put)]+, [Fe(ava)]+, and [Fe(bis)]+ that dominate at pH < 5 led to a forward metal-templated synthesis approach to generate the Fe(III)-loaded macrocycles, with apo-macrocycles furnished upon incubation with EDTA. This mini-review aims to capture the rich chemistry and chemical biology of these seemingly simple compounds.Graphical abstract