Abstract
Fragment-based screening methods can be used to discover novel
active site or allosteric inhibitors for therapeutic intervention. Using
saturation transfer difference (STD) NMR and in vitro activity assays,
we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase
(RT) with distinct chemical scaffolds and mechanisms compared
to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/
nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit
RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT
in the micromolar range while retaining potency against RT variants
carrying one of three major NNRTI resistance mutations: K103N,
Y181C, or G190A. These compounds also inhibit Moloney murine leukemia
virus RT but not the Klenow fragment of Escherichia coli DNA
polymerase I. Steady-state kinetic analyses demonstrate that one of
these fragments is a competitive inhibitor of HIV-1 RT with respect to
deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second
compound is a competitive inhibitor of RT polymerase activity
with respect to the DNA template/primer (T/P), and consequently also
inhibits RNase H activity. The dNTP competing RT inhibitor retains
activity against the NRTI-resistant mutants K65R and M184V, demonstrating
a drug resistance profile distinct from the nucleotide competing
RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-
6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing
compound inhibits HIV-1 replication at a step consistent with an RT
inhibitor. Screening of additional structurally related compounds to
the three fragments led to the discovery of molecules with improved
potency against HIV-1 RT. These fragment inhibitors represent previously
unidentified scaffolds for development of novel drugs for HIV-
1 prevention or treatment.
Project
