Selective Recognition of a Single HIV‑1 G‑Quadruplex by Ultrafast Small-Molecule Screening

G-quadruplexes (G4s)
are implicated in pathological processes such
as cancer and infective diseases. Their targeting with G4-ligands
has shown therapeutic capacity. Most of the current G4-ligands are
planar molecules, do not discriminate among G4s, and have poor druglike
properties. The available methods to identify compounds selective
for one single G4 are often time-consuming. Here, we describe the
development, validation, and application of an affinity-selection
mass spectrometry method that employs unlabeled G4 oligonucleotides
as targets and allows testing of up to 320 unmodified small molecules
in a single tube. As a proof of concept, this method was applied to
screen a library of 40 000 druglike molecules against two G4s,
transcriptional regulators of the HIV-1 LTR promoter. We identified
nonplanar pyrazolopyrimidines that selectively recognize and stabilize
the major HIV-1 LTR G4 possibly by fitting and binding through H-bonding
in its unique binding pocket. The compounds inhibit LTR promoter activity
and HIV-1 replication. We propose this method to prompt the fast development
of new G4-based therapeutics.