Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound , a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound is a<3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4-11 cells with an IC of 3 μM after 6 h.

Site-specific modification of proteins leads to invaluable structural and reactivity information for biochemical and medicinal studies and to new biotherapeutics. 1 Modification frequently depends on reaction of electrophilic reagents with nucleophilic atoms of the protein. 1 Classic examples include reaction of BrCN at S, 2a RNCS at N, 2b and electrophilic halogens at the tryptophan π-system. 2c The organic reagents which react with tryptophan or tyrosine react much more slowly or not at all with the less electron-rich phenylalanine. Moreover, the same electrophilic or oxidizing reagents also enter into unwanted side reactions with nucleophilic S-or N-donors. 1 Here we show that metal π-complexation to the arene-containing amino acid phenylalanine can succeed even in the face of S-and N-donor ligands found in methionine, cystine, or histidine, leaving these residues unchanged. Metal π-complexation to amino acids and dipeptides has been studied in nonaqueous solvents, 3 and metals have been covalently attached to proteins indirectly, by using organic ligands which are attached to mercapto or amino sites. 4 In contrast, here we report the attachment of a metal directly to an arene ring in a protein in water. Our strategy for reversible derivatization of aryl amino acids exploits the capability of CpRu + and Cp*Ru + fragments to bind to arenes, 3,5 providing air-stable products from which arene subsequently can be released on photolysis. We have made chelates 1, which unlike CpRu + and Cp*Ru + derivatives are new compounds featuring a bound amino ligand, 6 a nucleophile or water-solubilizing group locked by coordination until release by an incoming aryl amino acid. A series of experiments culminating with reaction of the small protein secretin at 10 -4 M concentrations demonstrate high arenophilicity and water tolerance in rapid room-temperature reactions, promising conditions for applications to proteins.Scheme 1 outlines the preparation of homologous chelates 1a,b. Without purification of intermediates, thallium salts 2 were formed in 56-60% overall yield from salts Br(CH 2 ) n NH 3 Br by monoalkylation of CpLi (2 equiv), 6d-8 protection of the amino nitrogen, and deprotonation of the resulting cyclopentadiene mixture using TlOEt. 7b Metathesis between 2 and [Cl(µ-Cl)Ru(η 6 -arene)] 2 and exchange to the noncoordinating PF 6 -anion 5a provided sandwich complexes 3, which were deprotected by hydrogenolysis. 5e,9 Photolysis of 4 in CH 3 CN produced 1. The rate of reaction was greater for the longer side chain (n ) 3) and for the smaller arene. 10a Conversely, arene complexation to 1 in CD 3 NO 2 is about 2 times faster for the shorter side chain, comparisons which may indicate that the chelate ring in 1a is more strained. 10b Arenes (Scheme 2) of widely disparate steric demand, such as 1,3-dimethylbenzene (5a) and 1,4-di-tert-butylbenzene (5b) readily opened chelate 1a under similar conditions (CD 3 NO 2 or CD 3 -OD, 60°C, 1-6 h), to give sandwich complexes 6. Amino acid derivatives 5c-h could also be used, ...