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[This corrects the article DOI: 10.1039/C9SC05586J.].

[This corrects the article DOI: 10.1039/C9SC05586J.].

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

Fragment-based drug discovery (FBDD) is a powerful strategy for the identification of new bioactive molecules. FBDD relies on fragment libraries, generally of modest size, but of high chemical diversity. Although good chemical diversity in FBDD libraries has been achieved in many respects, achieving shape diversity - particularly fragments with three-dimensional (3D) structures - has remained challenging. A recent analysis revealed that >75% of all conventional, organic fragments are predominantly 1D or 2D in shape. However, 3D fragments are desired because molecular shape is one of the most important factors in molecular recognition by a biomolecule. To address this challenge, the use of inert metal complexes, so-called 'metallofragments' (mFs), to construct a 3D fragment library is introduced. A modest library of 71 compounds has been prepared with rich shape diversity as gauged by normalized principle moment of inertia (PMI) analysis. PMI analysis shows that these metallofragments occupy an area of fragment space that is unique and highly underrepresented when compared to conventional organic fragment libraries that are comprised of orders of magnitude more molecules. The potential value of this metallofragment library is demonstrated by screening against several different types of proteins, including an antiviral, an antibacterial, and an anticancer target. The suitability of the metallofragments for future hit-to-lead development was validated through the determination of IC50 and thermal shift values for select fragments against several proteins. These findings demonstrate the utility of metallofragment libraries as a means of accessing underutilized 3D fragment space for FBDD against a variety of protein targets.

Fragment-based drug discovery (FBDD) is a powerful strategy for the identification of new bioactive molecules. FBDD relies on fragment libraries, generally of modest size, but of high chemical diversity. Although good chemical diversity in FBDD libraries has been achieved in many respects, achieving shape diversity - particularly fragments with three-dimensional (3D) structures - has remained challenging. A recent analysis revealed that >75% of all conventional, organic fragments are predominantly 1D or 2D in shape. However, 3D fragments are desired because molecular shape is one of the most important factors in molecular recognition by a biomolecule. To address this challenge, the use of inert metal complexes, so-called 'metallofragments' (mFs), to construct a 3D fragment library is introduced. A modest library of 71 compounds has been prepared with rich shape diversity as gauged by normalized principle moment of inertia (PMI) analysis. PMI analysis shows that these metallofragments occupy an area of fragment space that is unique and highly underrepresented when compared to conventional organic fragment libraries that are comprised of orders of magnitude more molecules. The potential value of this metallofragment library is demonstrated by screening against several different types of proteins, including an antiviral, an antibacterial, and an anticancer target. The suitability of the metallofragments for future hit-to-lead development was validated through the determination of IC50 and thermal shift values for select fragments against several proteins. These findings demonstrate the utility of metallofragment libraries as a means of accessing underutilized 3D fragment space for FBDD against a variety of protein targets.

Herein, we describe the synthesis and characterisation of four new supramolecular cobalt conjugates of antimicrobial peptides functionalised with terpyridine ligands (L). Peptides were chosen based on the well-established arginine-tryptophan (RW)3 motif, with terpyridine-derivatized lysine (Lys(tpy)) added to the sequence, or replacing tryptophan residues. Self-assembly of the antimicrobial peptides with Co(BF4)2·6H2O formed exclusively CoL2 dimers (for peptides with one tpy ligand each) and Co2L4 metallo-macrocycles (for peptides with two tpy ligands for each peptide), which could be 'locked' by oxidation of Co(+II) to Co(+III) with ammonium ceric nitrate. The Co-peptide complexes were characterised by mass spectrometry and in solution by NMR spectroscopy, including 2D diffusion ordered NMR spectroscopy (DOSY) which confirmed the proposed stoichiometries. The antimicrobial activity of the novel peptides and their metallo-supramolecular assemblies was investigated by determination of their minimal inhibitory concentration (MIC) against a panel of Gram-positive and Gram-negative bacteria. Complexation with cobalt increases the activity of the peptides in almost every case. Most of the new metal–peptide conjugates showed good activity against Gram-positive bacteria, including a multi-resistant S. aureus strain and the opportunistic pathogenic yeast C. albicans (down to 7 μmol l−1 for the most active Co2L4 derivate), a value that is increased five-fold compared to the lysine-derivatized peptide ligand alone. Interestingly, conjugates of the CoL2 type also showed decent activity against Gram-negative bacteria including the WHO-flagged problematic A. baumannii strain (down to 18 μmol l−1 for the most active derivative). [ABSTRACT FROM AUTHOR]

The synthesis and characterization of three compounds [Rh(η5‐C5Me5)Cl(N^N)]PF6 (N^N=4,4'‐disubstituted‐2,2'‐bipyridines, 1–3) are described. The cationic complexes contain the bidentate ligands N^N=4,4'‐di‐tert‐butyl‐2,2'‐bipyridine (1), N^N=4,4'‐dinonyl‐2,2'‐bipyridine (2) and N^N=4,4'‐diamino‐2,2'‐bipyridine (3). The complex salts were obtained by the bridge‐splitting reaction from the precursor [{Rh(η5‐C5Me5)(μ‐Cl)Cl}2] and subsequent salt metathesis affording their corresponding hexafluorido phosphate salts. All compounds were characterized by elemental analysis and spectroscopic means. Additionally, the molecular structure of compound 3 in the solid was determined by a single‐crystal X‐ray diffraction study. The cytotoxicity of all three compounds was examined by MTT assay against two cancer cell lines – HT‐29 (colon adenocarcinoma) and MCF‐7 (human breast adenocarcinoma) ‐ and normal human fibroblast cells (GM5657T). Compound 1 has moderate cytotoxicity against both cell lines, while compound 2 is seven to nine times more cytotoxic than cisplatin against MCF‐7 and HT‐29, respectively. In contrast to cisplatin, both compounds are more active against cancer cells than fibroblasts, thus showing some cancer selectivity. [ABSTRACT FROM AUTHOR]

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore‐type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well‐established arginine‐tryptophane combination K(RW)3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti‐bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a–c) ; (iii) the non‐siderophore chelators deferasirox (DFX) and deferiprone‐carboxylate (DFP‐COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR‐MS). 55Fe uptake assays indicate a receptor‐mediated uptake for 4 a–c, DFP‐COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron‐depleted medium. MIC values were as low as 2 μM (for P1‐DFP) on wild type P. aeruginosa. The activity of P1‐DFO and P1‐DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 μM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55Fe uptake studies, the P1‐DFX conjugate had excellent antibacterial activity compared to P1 (2 μM, and as low as 0.25 μM on a receptor‐deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1‐DFP and P1‐DFX being the most potent conjugates. [ABSTRACT FROM AUTHOR]

The synthesis and characterization of four compounds [M(η5‐C5Me5)(N^N)Cl]PF6 [N^N=4,7‐dichloro‐1,10‐phenanthroline with M=Rh, 1, and M=Ir, 2, and N^N=4'‐(4‐chlorophenyl)‐2,2':6',2''‐terpyridine in the κ2N,N'‐coordination mode with M=Rh, 3, and M=Ir, 4] are described. All compounds were characterized by spectroscopic means and their molecular structures in the crystal were confirmed by single‐crystal X‐ray diffraction studies. The cytotoxicity of all compounds was evaluated by MTT assay against the three cancer cell lines HeLa (cervical carcinoma), HT‐29 (colon adenocarcinoma) and MCF‐7 (human breast adenocarcinoma). The complexes 3 and 4 display promising activity with IC50 values of 1 μM. The rhodium(III) complex 1 also shows highly improved cytotoxicity compared to cisplatin against the cancer cell lines HT‐29 and MCF‐7. In contrast to this, the iridium(III) complex 2 is even less active against the HeLa cell line than cisplatin. [ABSTRACT FROM AUTHOR]

A simple and relatively inexpensive procedure for preparing a biologically active peptide using solid phase peptide synthesis (SPPS) is described. Fourth-year undergraduate students have gained firsthand experience from the solid-phase synthesis techniques and they have become familiar with modern analytical techniques based on the particular properties of the marker molecules, such as fluorescence spectra or electrochemistry.

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small‐molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore‐linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin‐based prodrugs were synthesised, and their catalyst‐mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore‐linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation. [ABSTRACT FROM AUTHOR]