2 research outputs found
Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity
Telomycin
(TEM) is a cyclic depsipeptide antibiotic active against Gram-positive
bacteria. In this study, five new natural telomycin analogues produced
by <i>Streptomyces canus</i> ATCC 12646 were identified.
To understand the biosynthetic machinery of telomycin and to generate
more analogues by pathway engineering, the TEM biosynthesis gene cluster
has been characterized from <i>S. canus</i> ATCC 12646:
it spans approximately 80.5 kb and consists of 34 genes encoding fatty
acid ligase, nonribosomal peptide synthetases (NRPSs), regulators,
transporters, and tailoring enzymes. The gene cluster was heterologously
expressed in <i>Streptomyces albus</i> J1074 setting the
stage for convenient biosynthetic engineering, mutasynthesis, and
production optimization. Moreover, in-frame deletions of one hydroxylase
and two P450 monooxygenase genes resulted in the production of novel
telomycin derivatives, revealing these genes to be responsible for
the specific modification by hydroxylation of three amino acids found
in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors
were identified when characterizing an unusual precursor deacylation
mechanism during telomycin maturation. By <i>in vivo</i> gene inactivation and <i>in vitro</i> biochemical characterization
of the recombinant enzyme Tem25, the maturation process was shown
to involve the cleavage of previously unknown telomycin precursor-lipopeptides,
to yield 6-methylheptanoic acid and telomycins. These lipopeptides
were isolated from an inactivation mutant of <i>tem25</i> encoding a (de)Āacylase, structurally elucidated, and then shown
to be deacylated by recombinant Tem25. The TEM precursor and several
semisynthetic lipopeptide TEM derivatives showed rapid bactericidal
killing and were active against several multidrug-resistant (MDR)
Gram-positive pathogens, opening the path to future chemical optimization
of telomycin for pharmaceutical application
Probing Factor Xa ProteināLigand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands
The accurate prediction of proteināligand binding
affinity
belongs to one of the central goals in computer-based drug design.
Molecular dynamics (MD)-based free energy calculations have become
increasingly popular in this respect due to their accuracy and solid
theoretical basis. Here, we present a combined study which encompasses
experimental and computational studies on two series of factor Xa
ligands, which enclose a broad chemical space including large modifications
of the central scaffold. Using this integrated approach, we identified
several new ligands with different heterocyclic scaffolds different
from the previously identified indole-2-carboxamides that show superior
or similar affinity. Furthermore, the so far underexplored terminal
alkyne moiety proved to be a suitable non-classical bioisosteric replacement
for the higher halogenāĻ aryl interactions. With this
challenging example, we demonstrated the ability of the MD-based non-equilibrium
free energy calculation approach for guiding crucial modifications
in the lead optimization process, such as scaffold replacement and
single-site modifications at molecular interaction hot spots