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Current Drug Discovery Technologies
ISSN: 1570-1638
Current Drug Discovery Technologies
Volume 3, Number 2, July 2006
Contents
The Nuclear Overhauser Effect in the Lead Identification
Process Pharmacophore Models Pp. 91-100
Marilisa Leone, Hudson H. Freeze, Chui Sien Chan and Maurizio
Pellecchia
[Abstract]
Mechanism Study of N-Dephenylation Mediated through
a N para-Hydroxy Metabolite Pp.
101-114
Jianyao Wang, William DeMaio, Appavu Chandrasekaran, Li
Shen, Alvin C. Bach II,JoAnn Scatina and Rasmy Talaat
[Abstract]
NMR-Based Quality Control Approach for the Identification
of False Positives and False Negatives in High Throughput
Screening Pp. 115-124
Claudio Dalvit, Dannica Caronni, Nicola Mongelli, Marina
Veronesi and Anna Vulpetti
[Abstract]
Novel Anti-bacterials Against MRSA: Synthesis
of Focussed Combinatorial Libraries of Tri-substituted 2(5H)-furanones
Pp. 125-134
Eric Lattmann, Nison Sattayasai, Carl S. Schwalbe, Suwanna
Niamsanit,David C. Billington, Pornthip Lattmann, Christopher
A. Langley,Harjit Singh and Simon Dunn
[Abstract]
Selective Targeting of Liposomes to Macrophages
Using a Ligand with High Affinity for the Macrophage Scavenger
Receptor Class A Pp. 135-144
Patrick C.N. Rensen, J.C. Emile Gras, Eva K. Lindfors,
Ko Willems van Dijk,J. Wouter Jukema, Theo J.C. van Berkel
and Erik A.L. Biessen
[Abstract]
Protein Conformational Diseases: From Mechanisms
to Drug Designs Pp. 145-153
Jin-Chung Lin and Hsuan-Liang Liu
[Abstract]
Abstracts
[Back to top]
The Nuclear Overhauser Effect in the Lead Identification
Process Pharmacophore Models
Marilisa Leone, Hudson H. Freeze, Chui Sien Chan and Maurizio
Pellecchia
In the past several years nuclear magnetic resonance (NMR)
spectroscopy has emerged as a valuable tool in the drug discovery
field. In such context, several NMR-based techniques have
been developed aimed at the identification and subsequent
optimization of novel binders for a given protein target.
Among the different NMR approaches, those relying on the transferred
Nuclear Overhauser Effect (tr-NOE) appear to be particularly
useful as in some instances, in addition to binding, tr-NOE
may provide also structural information on the binding mode
of a ligand. In the current work we will reiterate the basic
principles and applications that are related to measurements
of tr-NOEs. The tr-NOE can be applied as a screening tool
to recognize ligands for a given target protein in a mixture
of compounds or to identify pair of molecules that bind to
a protein simultaneously on adjacent sites (interligand NOEs).
Moreover, in the case of peptide-ligands, tr-NOEs furnish
intra-molecular distance constraints that can be used to determine
their bioactive conformation. Starting from the conformation
thus obtained, a pharmacophoric model can be derived and later
used to search within a 3D database of small molecules to
find new potentially active non-peptide compounds that fit
the pharmacophore. We will report examples of each of the
above mentioned strategies.
[Back to top]
Mechanism Study of N-Dephenylation Mediated through
a N para-Hydroxy Metabolite
Jianyao Wang, William DeMaio, Appavu Chandrasekaran, Li
Shen, Alvin C. Bach II,JoAnn Scatina and Rasmy Talaat
A P450 catalyzed N-para-hydroxy metabolite was suggested
to be a prerequisite for N-dephenylation occurrence. Although
two mechanisms have been proposed to describe this process
as a consequence of either a chemical degradation or P450
lead epoxidation of the hydroxy metabolite, direct evidence
has not been demonstrated.
In this study, we started with a novel technique using a dipeptide,
Lys-Phe, to trap the byproduct of N-dephenylation, a quinone-like
compound, forming a peptide adduct to facilitate LC/MS characterization.
N-dephenylation via chemical degradation was assessed
by LC/MS characterization of the resulting (Lys-Phe)2-quinone
from 4-hydroxyphenyl-2-naphthylamine following interaction
with Lys-Phe in pH 7.4 buffer. N-dephenylation mediated by
P450 catalysis proposed was investigated in N-para-hydroxy
benzodioxane derivative incubated with mouse liver microsomes
in the presence of Lys-Phe in 50/50 H216O/H218O.
LC/MS demonstrated that only one of two hydroxy oxygens in
the byproduct was exchanged with water and the MS signal intensity
of the 16O labeled peptide adduct was equal to
that of 18O labeled. These observations suggested
us that the origin of the oxygen in the byproduct was from
water only, not from O2. Therefore, it appears
that N-dephenylation occurs via a stepwise process,
namely the substrate is initially metabolized to a N-para-hydroxy
metabolite by P450, which was readily oxidized to a quinone
imine/iminium chemically or enzymatically, then hydrolyzed
resulting in N-dephenylation. However, in our studies, the
proposed P450 mechanism involving epoxidation of a N-para-hydroxy
metabolite was disproved.
[Back to top]
NMR-Based Quality Control Approach for the Identification
of False Positives and False Negatives in High Throughput
Screening
Claudio Dalvit, Dannica Caronni, Nicola Mongelli, Marina
Veronesi and Anna Vulpetti
The quality of the data generated in a high throughput screening
(HTS) run is fundamental for selecting bona fide inhibitors
and for ensuring the capture of the full richness of inhibitors
present in a chemical library. For this purpose a quality
control filter based on three one dimensional (1D) proton
NMR experiments is proposed. The approach called SPAM (Solubility,
Purity and Aggregation of the Molecule) Filter requires the
acquisition of a 1D reference spectrum, a WaterLOGSY spectrum
and/or a selective longitudinal relaxation filter spectrum
for the identified hits dissolved in aqueous solution and
in the presence of a water soluble reference molecule. This
palette of experiments permits the rapid characterization
of the identity, purity, solubility and aggregation state
of the active compound. This knowledge is crucial for deriving
accurate IC50 and Kl values of the inhibitors,
for identifying false negatives and for detecting promiscuous
inhibitors. Only compounds that pass through the SPAM Filter
can be considered as starting points for medicinal chemistry
efforts directed toward lead optimization. Examples of this
approach in the identification of false positives in a screening
run against the enzyme thymidine phosphorylase (TP) and the
rescue of a false negative in a screening run against the
Ser/Thr kinase AKT1 are presented.
[Back to top]
Novel Anti-bacterials Against MRSA: Synthesis of Focussed
Combinatorial Libraries of Tri-substituted 2(5H)-furanones
Eric Lattmann, Nison Sattayasai, Carl S. Schwalbe, Suwanna
Niamsanit,David C. Billington, Pornthip Lattmann, Christopher
A. Langley,Harjit Singh and Simon Dunn
Mucobromic and mucochloric acid were used as building blocks
for the construction of a chemical combinatorial library of
3,4,5-trisubstituted 2(5H)-furanones. With these 2 butenolide
building blocks, and eight alcohols a sublibrary of 16 dihalogenated
5-alkoxy-2(5H)-furanones was prepared. This sublibrary of
5-alkoxylated furanones was reacted with 16 amines generating
a full size focussed combinatorial library of 256 individual
compounds. This three dimensional combinatorial library of
3-halogen-4-amino-5-alkoxy-2(5H)-furanones was prepared around
the benzimidazolyl furanone lead structure by applying a solution
phase combinatorial chemistry concept. Typical representatives
of the library were purified and fully characterized and one
x-ray structures was recorded, additionally.
The 3-bromo-4-benzimizazolyl-5-methoxy-2(5H)furanone, Br-A-l,
showed an MIC of 8 µg/ml against the multiresistant
Staphylococcus aureus ( MRSA).
[Back to top]
Selective Targeting of Liposomes to Macrophages Using
a Ligand with High Affinity for the Macrophage Scavenger Receptor
Class A
Patrick C.N. Rensen, J.C. Emile Gras, Eva K. Lindfors,
Ko Willems van Dijk,J. Wouter Jukema, Theo J.C. van Berkel
and Erik A.L. Biessen
Macrophages play an important role in inflammatory processes
and are crucially involved in the onset and progression of
atherosclerosis and tumorigenesis. Therefore, macrophages
are regarded as an excellent target for therapeutic intervention.
Since the scavenger receptor class A (SRA) is highly expressed
on macrophages, we developed in the present study an SRA-specific
particulate drug carrier by providing phosphatidylcholine
liposomes with a targeting ligand for SRA. To enable firm
association with liposomes, the high-affinity SRA ligand decadeoxyguanine
was covalently attached via a linker to lithocholic
oleate (LCO-dA2dG10). Incorporation
of LCO-dA2dG10 into liposomes resulted
in an increased electronegative surface charge and a dramatically
enhanced serum clearance (t½<
2 min versus > 5 h). The LCO-dA2dG10-induced
liposome clearance was fully dependent on SRA, as the clearance
could be efficiently inhibited by the SRA competitor polyinosinic
acid. LCO-dA2dG10 enhanced the affinity
of liposomes for SRA in vivo selectively, since introduction
of overall or clustered negative charges by other modifications
(e.g. oxidation, inclusion of phosphatidylserine,
or exposure of glutamic acid residues) did not affect their
serum clearance substantially, albeit that these modifications
resulted in an at least equally high negative surface charge.
LCO-dA2dG10 also increased the association
of liposomes with RAW264.7 cells, resulting in an enhanced
intracellular delivery and bioactivity of encapsulated dexamethasone-phosphate.
Therefore, the SRA-specificity of LCO-dA2dG10-liposomes
may be applied for the specific delivery of drugs to macrophages,
which may be of therapeutic benefit in general inflammatory
disorders, atherosclerosis, and tumorigenesis.
[Back to top]
Protein Conformational Diseases: From Mechanisms to
Drug Designs
Jin-Chung Lin and Hsuan-Liang Liu
Amyloidosis comprises a group of diseases characterized by
the deposition of insoluble protein fibrils in specific organs
and includes several serious medical disorders, such as Alzheimer’s
disease, prion-associated transmissible spongiform encephalitis,
and type II diabetes. Despite the structural dissimilarity
between the soluble proteins and peptides, these fibrils exhibit
similar morphologies under electron microscopy with a characteristic
“cross β-sheet”
pattern examined by x-ray fiber diffraction experiments. Many
studies have revealed that each of these diseases is associated
to a specific protein that is partially unfolded, misfolded,
and aggregated. However, the detailed structures of the causative
agents and the toxicity mechanisms are less known. This review
summarizes recent studies in the conformational disorders
leading to aggregation; including which proteins potentially
cause conformational diseases, the aggregation mechanisms
of these proteins, and recent researches on the conformational
changes using advanced experiments or molecular dynamics simulations.
Finally, current drug designs towards these protein conformational
diseases are also discussed. It is believed that the advances
in basic understanding of the mechanisms of conformational
changes as well as biological functions of these proteins
will shed light on the development and design of potential
interfering compounds against amyloid formation associated
with protein conformational diseases.
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