In Silico Analysis of the Ga3+/Fe3+ Competition for Binding the Iron-Scavenging Siderophores of P. aeruginosa-Implementation of Three Gallium-Based Complexes in the "Trojan Horse" Antibacterial Strategy

Abstract

The emergence of multidrug-resistant (MDR) microorganisms combined with the ever-draining antibiotic pipeline poses a disturbing and immensely growing public health challenge that requires a multidisciplinary approach and the application of novel therapies aimed at unconventional targets and/or applying innovative drug formulations. Hence, bacterial iron acquisition systems and bacterial Fe^2+/3+-containing enzymes have been identified as a plausible target of great potential. The intriguing "Trojan horse" approach deprives microorganisms from the essential iron. Recently, gallium's potential in medicine as an iron mimicry species has attracted vast attention. Different Ga³⁺ formulations exhibit diverse effects upon entering the cell and thus supposedly have multiple targets. The aim of the current study is to specifically distinguish characteristics of great significance in regard to the initial gallium-based complex, allowing the alien cation to effectively compete with the native ferric ion for binding the siderophores pyochelin and pyoverdine secreted by the bacterium P. aeruginosa. Therefore, three gallium-based formulations were taken into consideration: the first-generation gallium nitrate, Ga(NO₃)₃, metabolized to Ga³⁺-hydrated forms, the second-generation gallium maltolate (tris(3-hydroxy-2-methyl-4-pyronato)gallium), and the experimentally proven Ga carrier in the bloodstream-the protein transferrin. We employed a reliable in silico approach based on DFT computations in order to understand the underlying biochemical processes that govern the Ga³⁺/Fe³⁺ rivalry for binding the two bacterial siderophores.

Keywords: DFT; ESKAPE; Ga3+/Fe3+ competition; Pseudomonas aeruginosa; maltolate; pyochelin; pyoverdine; siderophore; transferrin.

Figure 1

Iron up-take systems in P. aeruginosa in correspondence to the scope of the present research, and possible ways for gallium’s interference according to its initial form: Pathway 1 represents the iron-siderophore access: (1A) The Fe-PCH complex is recognized and further internalized into the cell by the FptA receptor, a specific TonB-dependent outer membrane transporter. A fraction of Fe-PCH dissociates in the periplasm through an unknown mechanism, and the free ferric/ferrous ions are further transferred into the bacterial cytoplasm across the inner membrane via the PchH-PchI pathway, while another fraction of the complex is carried by the FptX into the cytoplasm; (1B) The Fe-PVD complex is imported into the cell through FpvA, a specific TonB-dependent membrane protein transporter for iron-loaded pyoverdines. Following the conformational changes in the FpvA transporter, the ferric ion is released through a still unknown process but most probably involving reduction of the metal without degradation or chemical modification of PVD. The FpvCDEF ABC transporter is expected to participate in the dissociation of iron. Additionally, the PvdRT-OpmQ pump in P. aeruginosa effluxes back the ‘bare’ siderophore or other metal-PVD complexes into the intracellular space. Pathway 2 depicts the Heme-internalization system typical for Gram–negative bacteria, the HasA-type hemophores, capable of acquiring Heme from a broad range of host hemoproteins, including hemoglobin, hemopexin, and myoglobin. Although the step-by-step process is still under debate, it is widely accepted that HasA acquires Heme without direct interaction with the hemoproteins of the invaded host. Pathway 3 illustrates the Fur-regulated bipartite system consisting of an outer membrane receptor protein, TbpA, and a surface-associated lipoprotein, TbpB, both binding transferrin in its holo-form (metal-loaded), while TbpA is able to bind the apo-form as well. In contrast to Pathways 1 and 2, ferric ions must first be liberated from transferrin prior to uptake by TbpA. This figure is created in accordance with data provided in Ref. [61].

Figure 2

Chemical structures of (A) the two iron-scavenging P. aeruginosa siderophores pyochelin (denoted as PCH) and pyoverdine (denoted as PVD). The metal-ligating groups are colored in pink; (B) the two generations of gallium(III)-based potential drugs, gallium nitrate (GaN) and gallium maltolate (GaM), and the Ga³⁺-carrier in the bloodstream, transferrin, with its metal-binding groups.

Figure 3

B3LYP optimized structures of Fe³⁺/Ga³⁺-bound ligands from the protein-buried (ε ≈ 4) active center of transferrin and Gibbs energies in kcal mol⁻¹ of metal substitution calculated at different theoretical levels in comparison with those experimentally observed and reported in Ref. [45]. The presented color scheme is further applied in Figures 4 to 7.

Figure 4

Gibbs energies of metal substitution (in kcal mol⁻¹) in four environments of different polarity and acidic pH ≈ 5 in correspondence to the pKa values of the hydrated metal structures optimized at the B3LYP/6-31+G(3d,p) level in [M_PCH_2OH]⁻ (A), [M_PCH_2W]⁺ (B), [M_2PCH]⁻ (C), and [M_PVD]⁻ (D). The applied color scheme has been previously presented in Figure 3.

Figure 5

Gibbs energies of metal substitution (in kcal mol⁻¹) in four environments of different polarity and physiological pH ≈ 7 in correspondence to the pKa values of the hydrated metal structures optimized at the B3LYP/6-31+G(3d,p) level in [M_PCH_2OH]⁻ (A), [M_PCH_2W]⁺ (B), [M_2PCH]⁻ (C), and [M_PVD]⁻ (D). The applied color scheme has been previously presented in Figure 3.

Figure 6

Gibbs energies of metal substitution (in kcal mol⁻¹) in four environments of different polarity with an initial gallium-based complex gallium(III) maltolate optimized at the B3LYP/6-31+G(3d,p) level in [M_PCH_2OH]⁻ (A), [M_PCH_2W]⁺ (B), [M_2PCH]⁻ (C), and [M_PVD]⁻ (D). The presented color scheme has been previously presented in Figure 3.

Figure 7

Gibbs energies of metal substitution (in kcal mol⁻¹) in four environments of different polarity with an initial gallium-based complex corresponding to the metal binding ligands in the active center of transferrin denoted as [Ga_Tf]²⁻ (explicit structure presented in Figure S1B) optimized at the B3LYP/6-31+G(3d,p) level in [M_PCH_2OH]⁻ (A), [M_PCH_2W]⁺ (B), [M_2PCH]⁻ (C), and [M_PVD]⁻ (D). The applied color scheme has been previously presented in Figure 3.