download report - Istituto Pasteur

P a r t i c i p a n t s :
Roberta Costi, professor; Gaetano Miele, post-doc fellow;
Giuliana Cuzzucoli Crucitti, PhD student; Federica Rosi,
Alberto Iacovo, graduate students, Giovanni Santilli, technician.
C o l l a b o r a t i o n s :
Università di Napoli “Federico II” (Prof. Ettore Novellino, Dr.
Luciana Marinelli); Università di Cagliari (Prof. Enzo
Tramontano); NCI at Bethesda, NIH, Bethesda, USA (Prof. Yves
Pommier); Katholieke Universiteit Leuven, Belgium (Prof.
Christophe Pannecouque); Politechnika Lodzka, Poland (Prof.
Grzegorz Bujacz).
Report of activity
The HIV-1 RT-associated RNase H function is a validated
and very attractive new target for HIV/AIDS
drug development (De Clercq, J Med Chem. 2005,
48:1297-1313; Tramontano, Mini Rev. Med Chem.
2006, 6:727-37; Himmel et al., ACS Chem Biol. 2006,
1:702-12); up to today no drug against the HIV-1 RTassociated
RNase H is: i) approved for therapy, ii)
under evaluation in clinical trial, iii) under later
stages of pre-clinical evaluation. The current available
information on the 3D structure of the HIV-1
RT (comprising its RNase H domain) give a solid
support for drug development by both in silico
screening and lead compound optimization through
docking studies and the first crystal structure of the
HIV-1 RT with an inhibitor bound to the RNase H
domain has been very recently reported by an
American academic team (Himmel et al., ACS Chem
Biol. 2006, 1:702-12).
Recently, two interesting DKA derivatives have been
reported. The first derivative, 4-[5-(benzoylamino)
thien-2-yl]-2,4-dioxobutanoic acid (BTDBA), originally
synthesized for HIV-1 IN inhibition, has been
shown to inhibit the HIV-1 RT RNase H function
without affecting its polymerase activity (Beutler et
al., PCT Int. Appl. 2006, 2006026619 A2). BTDBA
117
New antimicrobial and antiviral agents - AREA 6
Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents
targeted to the ribonuclease H function of the HIV-1 reverse
transcriptase enzyme
Principal investigator: Roberto Di Santo
Professor of Farmaceutical Chemistry and Toxicology
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49913150; Fax: (+39) 06 491491
roberto.disanto@uniroma1.it
provided the proof of concept for direct inhibition of
the HIV-1 RT RNase H associated activity by DKAs,
even though it was not highly selective for RNase H
since i) it inhibited in the same concentration range
also the HIV-1 IN in enzyme assays and ii) it did not
block the viral replication in cell-based assays.
The second DKA derivative, 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5hexenoic
acid ethyl ester (RDS1643) was recently
reported by our research group. In enzyme assays,
RDS1643 inhibited the HIV-1 RNase H activity with
an IC 50 value of 13 µM, it did not affect neither the
HIV-1 RDDP function nor the AMV and E. coli
RNase H activity, while it slightly inhibited the HIV-
1 IN reaction (IC 50 value of 92-98 µM)
(Tramontano et al., Antiv. Res. 2005, 65:117-24).
Noteworthy, in cell-based assays it was able to block
the replication of wild type HIV-1, showing an EC 50
value of 13 µM and a CC 50 value of 63 µM, and the
replication of three HIV-1 non-nucleoside RT
inhibitor (NNRTI) resistant viral mutants
(RT mutations were Y181C; K103N/Y181C;
K103R/V179D/P225H) showing EC 50 values of 7-
19 µM (Tramontano et al., Antiv. Res. 2005, 65:117-
24). Mode of action studies demonstrated that the
RDS1643 maximum adsorbance shifted in the presence
of the Mg 2+ ions. This results suggested that,
similarly to BTDBA (Mizrahi et al., J. Biol. Chem.
1994, 269: 19245-49), RDS1643 may sequestrate the
active site divalent metals having a specific binding
site on the HIV-1 RNase H domain (Tramontano et
al., Antiv. Res. 2005, 65:117-24).
More recently, BTDBA has been modeled into the
HIV-1 RNase H active site assuming that the DKA
triple-oxygen motif may interact with the protein
active site metal ions (Klumpp & Mirzadegan, Curr.
Pharm. Des. 2006, 12:1909-22). According to this
model, its aromatic moiety may extend towards the
W266, L422 and W426 amino acid residues on the
p51 subunit. Consistently, further modeling studies
proposed that RDS1643 may bind to the HIV-1 RNase