Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Medical Microbiology Section
Zusammenfassung
Die Schlüsselenzyme im Replikationskomplex aller
Vertreter der Flaviviridae sind die RNA NTPase/Helikase
und die RNA-abhängige RNA-Polymerase
(RdRp). Sie sind auch im Genom des Hepatitis C Virus
(HCV) kodiert. Die NTPase /Helikase löst enzymatisch
die doppelsträngige RNA-Struktur, indem
sie die Wasserstoffbrückenbindungen zwischen den
beiden Strängen aufbricht. Die dabei entstehenden
Einzelstränge sind das Substrat für die RdRp. Die
Helikase-Aktivität des Enzyms wird angetrieben
durch die Energie, die bei der NTP-Hydrolyse freigesetzt
wird. Daher können potenzielle Inhibitoren der
flaviviralen NTPase/Helikase an der Kopplung zwischen
NTP-Hydrolyse und Entwindung der RNA-
Stränge wirken, indem sie eine allosterische Nucleosid-Bindungsstelle
des Enzyms besetzen. Das zweite
Ziel für die antivirale Therapie ist die RdRp-Aktivität.
Wir stellen eine Familie von Nucleosid-Analoga vor
(Benzoxazole und Benzotriazole), die natürlich vorkommenden
Purinen ähneln. Die Substitution des
Benzoyl-Ringes mit Bromin-Atomen in Kombination
mit einer aliphatischen Kette in der Postion 1 oder 2
des Imidazol-Ringes führt zu einer erheblichen inhibitorischen
Wirkung dieser Verbindungen auf die
Helikase-Aktivität der HCV NTPase/Helikase und
RdRp. Um den Mechanismus der Inhibitorwirkungen
aufzuklären, wurde eine Ko-Kristallisierungsstudie
(Enzyme plus Inhibitor) durchgeführt.
Summary
RNA NTPase/helicase and RNA dependent RNA polymerase
(RdRp) represent the key enzymes of the replication
complex of all members of the Flaviviridae family.
The enzymes have been also encoded by viral genome
of the hepatitis C virus (HCV). The NTPase/helicase
is capable of enzymatically unwinding duplex RNA
structures by disrupting the hydrogen bonds that keep
the two strands together. The resulting single strands
serve as substrates for RdRp. The helicase activity is
dependent on the energy produced in course of the
NTP hydrolysis. Thus, potential specific inhibitors of
NTPase/helicase of Flaviviridae could act by inhibition
of the coupling of NTP hydrolysis to unwinding reaction
resulting from an occupation of a nucleoside-binding allosteric
site of the enzyme. The second target for antiviral
therapy is the activity of the RdRp. We present a
family of nucleoside base analogues (benzoxazole
and benzotriazole) that resemble natural purines. The
HCV-Inhibitors
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substitution of the benzoyl ring with bromine atoms
combined with aliphatic chain in the position 1 or 2 of
the imidazole ring generates considerable inhibitory
potential of the compounds towards the helicase activity
of the HCV NTPase/helicase and RdRp. To clear the
mechanism of action of the compounds a co crystallization
study (enzymes plus inhibitor) was initiated.
Introduction
Between the structural and nonstructural proteins of
HCV the enzymes of the replication complex appear to
be the most promising target for antiviral agents because
of the multiple enzymatic activities that are essential
for virus replication. Thus, compounds targeting
the enzymes may exert an synergistic antiviral effect
and therefore represent candidates for potent antiviral
drug.
Project Rescription and Results
Our previous screening of chemical libraries combined
with studies with RING-expanded nucleosides and with
derivatives of benzimidazoles and benzotriazoles indicated
that the extension of the benzoyl ring of the purine
base derivatives results in enhancing of inhibitory
activity of the compounds against helicase and/or polymerase
activities of Flaviviridae NTPase/helicases or
RdRp. We have designed and screened a broad range
of halogenized derivatives based on the structural skeleton
of a benzimidazole and/or benzotriazole. The modifications
included also addition of aliphatic residues
and ribose moiety as presented in the Figure 1.
Figure 1
Systematic screening of the compounds obtained combined
with rational substitutions revealed following features
of the derivatives of benzimidazoles and benzotriazoles
that appear to be conditio sine qua non for
their inhibiting properties. I) extended benzoyl ring (by
multiple substitutions with bromium atoms) and II) short
aliphatic chain (2-4 carbon atoms) placed in the position
1 or 2 of the imidazole ring. By a combination of
the modifications highly selective - in respect to the viral
enzymes inhibitors were obtained.