3 research outputs found
Probing the ATP Hydrolysis Cycle of the ABC Multidrug Transporter LmrA by Pulsed EPR Spectroscopy
Members of the ATP binding cassette (ABC) transporter
superfamily
translocate various types of molecules across the membrane at the
expense of ATP. This requires cycling through a number of catalytic
states. Here, we report conformational changes throughout the catalytic
cycle of LmrA, a homodimeric multidrug ABC transporter from <i>L. lactis.</i> Using site-directed spin labeling and pulsed
electronâelectron double resonance (PELDOR/DEER) spectroscopy,
we have probed the reorientation of the nucleotide binding domains
and transmembrane helix 6 which is of particular relevance to drug
binding and part of the dimerization interface. Our data show that
LmrA samples a very large conformational space in its apo state, which
is significantly reduced upon nucleotide binding. ATP binding but
not hydrolysis is required to trigger this conformational change,
which results in a relatively fixed orientation of both the nucleotide
binding domains and transmembrane helices 6. This orientation is maintained
throughout the ATP hydrolysis cycle until the protein cycles back
to its apo state. Our data present strong evidence that switching
between two dynamically and structurally distinct states is required
for substrate translocation
Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors
We propose a structure-based
protocol for the development of customized
covalent inhibitors. Starting from a known inhibitor, in the first
and second steps appropriate substituents of the warhead are selected
on the basis of quantum mechanical (QM) computations and hybrid approaches
combining QM with molecular mechanics (QM/MM). In the third step the
recognition unit is optimized using docking approaches for the noncovalent
complex. These predictions are finally verified by QM/MM or molecular
dynamic simulations. The applicability of our approach is successfully
demonstrated by the design of reversible covalent vinylsulfone-based
inhibitors for rhodesain. The examples show that our approach is sufficiently
accurate to identify compounds with the desired properties but also
to exclude nonpromising ones
Development of Novel Peptide-Based Michael Acceptors Targeting Rhodesain and Falcipainâ2 for the Treatment of Neglected Tropical Diseases (NTDs)
This paper describes
the development of a class of peptide-based
inhibitors as novel antitrypanosomal and antimalarial agents. The
inhibitors are based on a characteristic peptide sequence for the
inhibition of the cysteine proteases rhodesain of Trypanosoma
brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity
of novel unsaturated electrophilic functions such as vinyl-sulfones,
-ketones, -esters, and -nitriles. The Michael acceptors inhibited
both rhodesain and falcipain-2, at nanomolar and micromolar levels,
respectively. In particular, the vinyl ketone <b>3b</b> has
emerged as a potent rhodesain inhibitor (<i>k</i><sub>2nd</sub> = 67 Ă 10<sup>6</sup> M<sup>â1</sup> min<sup>â1</sup>), endowed with a picomolar binding affinity (<i>K</i><sub>i</sub> = 38 pM), coupled with a single-digit micromolar activity
against Trypanosoma brucei brucei (EC<sub>50</sub> = 2.97 ÎŒM), thus being considered as a novel lead
compound for the discovery of novel effective antitrypanosomal agents