Potent 1,2,4-Triazino[5,6 b]indole-3-thioether Inhibitors of the Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis.

A common cause of resistance to kanamycin (KAN) in tuberculosis is overexpression of the enhanced intracellular survival (Eis) protein. Eis is an acetyltransferase that multiacetylates KAN and other aminoglycosides, rendering them unable to bind the bacterial ribosome. By high-throughput screening, a series of substituted 1,2,4-triazino[5,6 b]indole-3-thioether molecules were identified as effective Eis inhibitors. Herein, we purchased 17 and synthesized 22 new compounds, evaluated their potency, and characterized their steady-state kinetics. Four inhibitors were found not only to inhibit Eis in vitro, but also to act as adjuvants of KAN and partially restore KAN sensitivity in a Mycobacterium tuberculosis KAN-resistant strain in which Eis is upregulated. A crystal structure of Eis in complex with a potent inhibitor and CoA shows that the inhibitors bind in the aminoglycoside binding site snugly inserted into a hydrophobic cavity. These inhibitors will undergo preclinical development as novel KAN adjuvant therapies to treat KAN-resistant tuberculosis.

Combating Enhanced Intracellular Survival (Eis)-Mediated Kanamycin Resistance of Mycobacterium tuberculosis by Novel Pyrrolo[1,5-a]pyrazine-Based Eis Inhibitors.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure-activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.

Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis.

A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.

Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.