High-resolution Crystallographic Analysis of the Competitive Binding of a Novel Nucleotide Analog GS-9148-Diphosphate to HIV-1 Reverse Transcriptase
Eric Lansdon*, D Samuel, L Lagpacan, K White, C Boojamra, R Mackman, T Cihlar, A Ray, M McGrath, and S Swaminathan
Gilead Sci, Foster City, CA, US
Background: GS-9148 is a phosphonate analog of
2’-deoxyadenosine monophosphate (dAMP) and is orally administered as its
lymphoid-targeted amidate prodrug GS-9131. GS-9148 has a unique resistance
profile including maintained activity against nucleoside reverse transcriptase
(RT) inhibitor resistance mutations M184V, K65R, L74V, and multiple thymidine
analog mutations. To determine the mechanism of potent HIV RT inhibition by
GS-9148-diphosphate (DP) and to gain insight into its maintained activity
towards resistant mutants, crystal structures of HIV RT bound to different
substrates were determined.
Methods: HIV-1 RT was co-crystallized with
co-valently tethered double stranded DNA. The primer strand DNA was terminated
with ddCMP allowing the next nucleotide to bind in the polymerase active site.
A crystal structure was determined with the binary RT-DNA complex to 3.1A
resolution. Crystals of this complex were soaked with GS-9148-DP and dATP to
obtain structures at 2.7 and 2.9A, respectively.
Results: The structure of the RT-DNA complex showed
that the fingers loop is in an open conformation poised for dNTP binding.
Binding of either GS-9148-DP or dATP resulted in a conformational change
including the closing of the fingers domain. Both substrates bound in a similar
fashion establishing Watson-Crick base pairs with the templating dTMP. R72
interacts with the α- and β-phosphates for both nucleotides and a
coordinated Mg2+ ion is present in both structures. K65, K70, and
K219 also interacted similarly with the phosphates of both nucleotides. One
minor difference was the R72 interaction with the phosphonate oxygen of
GS-9148-DP versus the α-phosphate oxygen of dATP. Although the 2’-fluoro
of GS-9148-DP was in close proximity to Q151 (2.9A), there were minimal changes
in the position of Q151 in the 2 ternary structures. Other residues whose mutations
are known to confer NRTI resistance such as M184, Y115, and L74 show no marked
differences between both structures.
Conclusions: The crystal structure of GS-9148-DP
bound to RT-DNA complex is the highest resolution ternary RT structure with a
nucleotide inhibitor. The ability of GS-9148-DP to mimic the active site
contacts of dATP explains its effective inhibition of wild type RT and potent
activity against common RT resistance mutations. The close proximity of the
2’-fluoro may explain why Q151M was the only mutation that conferred moderate
in vitro resistance in patient isolates.