| Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 8, Number 1, February 2005
Contents
Streamlining the Discovery of Effective Anti-Malarial
Agents
Guest Editor: Norman C. Waters
Editorial from Editor-in-Chief
Richard B. van Breemen
[Editorial
In PDF]
Editorial from Guest Editor
Norman C. Waters
[Editorial
In PDF]
Structure-Based Drug Discovery for Plasmodium
falciparum Pp.5-14
Christopher Mehlin
[Abstract] [Full
text article]
Fatty Acid Synthesis as a Target for Antimalarial
Drug Discovery Pp.15-26
Jeff Zhiqiang Lu, Patricia J. Lee, Norman C. Waters and
Sean T. Prigge
[Abstract] [Full
text article]
Rational Inhibitor Design and Iterative Screening
in the Identification of Selective Plasmodial Cyclin Dependent
Kinase Inhibitors Pp.27-38
Susan M. Keenan, Jeanne A. Geyer, William J. Welsh, Sean
T. Prigge and Norman C. Waters
[Abstract] [Full
text article]
1,4-Bis(3-Aminopropyl)Piperazine Libraries: From
the Discovery of Classical Chloroquine-Like Antimalarials
to the Identification of New Targets Pp.39-48
Rebecca Deprez-Poulain and Patricia Melnyk
[Abstract] [Full
text article]
Dual Molecules as New Antimalarials Pp.49-62
Xavier J. Salom-Roig, Abdallah Hamze, Michele Calas and
Henri J. Vial
[Abstract] [Full
text article]
Targeting the Hemozoin Synthesis Pathway for New
Antimalarial Drug Discovery: Technologies for In Vitro
β-Hematin Formation Assay Pp.63-79
Babu L. Tekwani and Larry A. Walker
[Abstract] [Full
text article]
The New Permeability Pathways: Targets and Selective
Routes for the Development of New Antimalarial Agents Pp.81-88
Henry M. Staines, J. Clive Ellory and Kelly Chibale
[Abstract] [Full
text article]
The Role of In Vitro ADME Assays in Antimalarial
Drug Discovery and Development Pp.89-98
Todd W. Shearer, Kirsten S. Smith, Damaris Diaz, Constance
Asher and Julio Ramirez
[Abstract] [Full
text article]
Abstracts
[Back to top]
Editorial from Editor-in-Chief
Richard B. van Breemen
[Editorial
In PDF]
Combinatorial Chemistry & High Throughput Screening
begins its eighth year of publication with this issue. Given
this perspective, it is interesting how rapidly this field
is changing and yet still remains relevant to the discovery
and development of new drugs, catalysts, and other materials.
For example, in just eight years we have witnessed a trend
away from the practice of assembling enormous random libraries
of compounds for drug discovery. Instead, libraries of “drug
like” compounds are being assembled for screening that
exclude compounds with undesirable physical properties such
as excessively high molecular weight that prevents absorption
following oral administration or poor solubility that impedes
formulation and screening. In combinatorial chemistry, the
new approach of diversity oriented synthesis is being used
to generate a greater variety of compounds for screening than
ever before so that the synthetic process may become more
efficient at generating chemical diversity. The need for more
diversity in screening programs has also renewed interests
in natural products as sources of diverse chemical structures.
Despite the demand for greater diversity in early screening
programs, the synthesis of structurally related compounds
from a particular scaffold remains popular after a lead compound
has been identified that may serve as a model. Finally, in
an effort to enhance the productivity of high throughput screening
programs for drug discovery, drug development assays are being
incorporated earlier than ever in the discovery phase in a
form of high throughput screening that is being called high
content screening.
During 2005 Combinatorial Chemistry & High Throughput
Screening will continue to publish review articles and
original research papers in all areas of combinatorial chemistry
and high throughput screening. Regular issues will be alternated
with special issues that contain a collection of review and
research papers focusing on a single topic of current interest.
Some of the special issues will be organized by members of
our Editorial Board whereas others will be organized by guest
editors. For example, this first issue of 2005 has been put
together by guest editor Norman C. Waters of the Walter Reed
Army Institute of Research and concerns the discovery of new
drugs for the important disease malaria. The next issue of
CCHTS will be devoted to regular articles. Whether contributed
by authors for a regular issue or as part of a special issue
on a hot topic, all papers appearing in this journal will
continue to be peer-reviewed.
During 2005 eight issues of CCHTS are planned, and
this frequency of publication remains the highest in the field
of combinatorial chemistry or high throughput screening. Papers
published in this journal are abstracted and indexed by the
major services including BIOSIS, Chemical Abstracts, Current
Contents/Life Sciences, EMBASE, BIOBASE, Science Citation
Index-Expanded, Index Medicus/MEDLINE, and CAB Abstracts.
In addition, the impact factor of Combinatorial Chemistry
High Throughput Screening increased to 2.53 this year, which
is its highest level ever according to the ISI Journal Citation
reports. Therefore, papers published in CCHTS are highly visible
to the research community.
The homepage of our journal and abstracts of articles may
be found at the following Internet address: http://www.bentham.org/cchts.
Information for authors may also be found at our website.
Authors will be pleased to learn that we accept manuscripts
in either paper or electronic format, and our readers and
subscribers will continue be able to obtain CCHTS in printed
or electronic format. Through a combination of frequent publication
and high visibility, Combinatorial Chemistry & High Throughput
Screening remains a unique and essential scientific journal
defining the intersection of these two interdependent disciplines.
I would like to thank the distinguished members of our Editorial
Board, our Regional Editors, our able Guest Editors, the authors
who contributed reviews and research papers, and of course
you, our readers, for the continuing success of our journal.
[Back to top]
Editorial from Guest Editor
Norman C. Waters
[Editorial
In PDF]
Malaria continues to kill at an alarming rate of about 1-2
million deaths annually. Early predictors have health officials
extremely concerned because it is estimated that this death
rate will increase due to the high level of malaria drug resistance
and the lack of effective and inexpensive chemotherapy options.
Decades of malaria drug development have resulted in structurally
similar classes of compounds that do little to circumvent
established drug resistance mechanisms in the parasite. In
addition to drug resistance, the mechanisms of antimalarial
activity of several of these drugs are unknown or not very
well understood which complicates efforts to design effective
derivatives of these drugs. Knowledge-based drug design is
needed more than ever in the fight against malaria to introduce
new drugs with known mechanisms of actions. Structure-based
approaches to malaria drug discovery to include high throughput
screening, virtual and computer-aided design, and synthesis
of chemical libraries, are currently being applied to develop
the next generation of effective antimalarials. In this age
of significant drug resistance, these approaches provide an
opportunity to introduce novel chemical entities into the
malaria drug development pipeline.
In this issue of Combinatorial Chemistry & High Throughput
Screening, we have assembled a collection of articles
on the discovery of new antimalarial agents. This issue will
focus on strategies of knowledge-based drug design to identify
new chemical entities, in addition to topics on target screens
and library design.
We begin the theme with an article by C. Mehlin on the efforts
to gain structural information on malarial proteins. This
review focuses on several malarial enzyme targets for which
there are crystal structures available. The difficulty associated
with the expression and purification of malarial proteins
is a major stumbling block in rational drug design and these
setbacks are discussed. For those enzymes that are amenable
to structural determination, co-crystallization of inhibitors
with enzymes provides a wealth of information required to
guide antimalarial drug discovery.
Although rational drug design methodologies are being applied
to several malaria enzymes, we chose to review efforts on
two recently developed malaria drug target programs. Zhiqiang
and coworkers compare fatty acid synthesis between bacteria
and malaria and describe recent efforts to target these unique
enzymes in Plasmodium falciparum. In particular,
targeting β-ketoacyl-ACP
synthase III (KASIII) to identify potent antimalarial agents
is presented. Continuing with this theme, Keenan and coworkers
describe an iterative process that includes high throughput
screens and computer aided inhibitor design to select potent,
yet specific inhibitors of the malarial cyclin dependent protein
kinases (CDKs). Unlike fatty acid biosynthesis, CDKs are highly
conserved throughout eukaryotes and this article provides
an overview of the approaches used to target conserved enzymes
that are essential for malaria growth and development.
Targeting malarial enzymes or metabolic pathways for chemotherapeutic
development requires synthesis of specific chemical libraries.
Initial drug discovery leads may arise from the screening
of chemical databases; however, refinement towards specificity
and potency must be supported by synthetic efforts. Deprez-Poulain
and Melnyk describe the synthesis of piperazine libraries
with potent antimalarial activity. They then describe how
these libraries were used to elicit a possible mechanism of
action similar to that of chloroquine and expand on the approach
with the identification of the aminopeptidase Pfa-M1 as a
potential target of these compounds.
In addition to specific targets, metabolic pathways can be
targeted to kill the malaria parasite. Three articles in this
series present evidence that compounds can be designed to
inhibit the metabolic processes within the parasite. Salom-Roig
and coworkers describe the synthesis of compounds that appear
to have dual functions associated with antimalarial activity.
Three generations of compounds, (bis-quaternary salts, bis-amidines,
and bis-thiazolium salts), were synthesized to target phosphatidylcholine
biosynthesis and heme detoxification pathways in the parasite.
These compounds have potent activity against malaria parasites
in culture and in animal models. Tekwani and Walker continue
the theme of targeting heme detoxification and discuss several
in vitro b-hematin formation assays. The development of these
assays has made it possible to screen for inhibitors of hemozoin
synthesis in high throughput formats. Several chemical classes
have been screened in this system to include quinolines, xanthones,
azoles, and natural products. The last article on targeting
systems rather than individual targets is from Staines and
coworkers. They describe exploitation of the new permeability
pathways (NPP) in the parasite using two novel approaches.
In the first, they describe several chemical classes that
are effective inhibitors this permeability pathway which is
lethal in the parasite. Second, they describe the use of this
pathway to deliver drugs or inhibitory compounds into the
parasite. Finding novel ways of introducing antimalarial agents
into the parasite is a challenge, since malaria is an intracellular
parasite enveloped within several biological membranes. The
NPP may provide a way of ensuring that particular compounds
are delivered to the specified target within the parasite.
We conclude with a topic that many investigators do not like
to think about because it can mean the end to any potential
lead compound from their respective drug discovery programs:
pharmacokinetics and toxicity. Shearer and coworkers describe
the role of metabolic studies in the development of new antimalarial
agents. In particular, they describe ADME (absorption, distribution,
metabolism, and excretion) assays that if used properly within
the drug discovery pipeline, can effectively guide antimalarial
drug design and prevent many setbacks that cost a significant
amount of time and money.
In summary, we have collected several articles that discuss
approaches to antimalarial drug discovery using rational drug
design methodologies. Although there are multiple efforts
in malaria drug discovery, these articles deal with new areas
that have the potential of introducing new chemical entities
into the malaria drug development pipeline. It is hoped that
this volume will provide insight into the rational drug design
paradigm and how it can be applied for the discovery of novel
antimalarial agents. For scientist working outside the realm
of tropical diseases, this issue may serve as a reminder that
malaria remains a significant problem in the world and that
every effort is essential to keep the malaria drug development
pipeline full with the next generation of potential malaria
chemotherapeutics.
[Back to top]
Structure-Based Drug Discovery for Plasmodium falciparum
Christopher Mehlin
[Full text
article]
X-ray crystallography is a technique which is finding increasing
utility in the effort to find new antimalarial drugs. This
is in spite of the serious difficulties often encountered
in obtaining sufficient quantities of protein to crystallize.
This review provides an overview of the Plasmodium falciparum
proteins which have been crystallized with bound inhibitors
and the methodology employed in the heterologous expression
of these proteins. Lactate dehydrogenase, plasmepsin II, and
triosphosphate isomerase are the most advanced targets of
structure-based drug design, but nine other P. falciparum
proteins have been crystallized with inhibitors as well, and
this is clearly an area which is moving very quickly. Some
consideration will also be given to the limitations of structure-based
drug discovery with respect to known antimalarial drugs.
[Back to top]
Fatty Acid Synthesis as a Target for Antimalarial Drug Discovery
Jeff Zhiqiang Lu, Patricia J. Lee, Norman C.
Waters and Sean T. Prigge
[Full text
article]
In biological systems, fatty acids can be synthesized by
two related, but distinct de novo fatty acid synthase
(FAS) pathways. Human cells rely on a type I FAS whereas plants,
bacteria and other microorganisms contain type II FAS pathways.
This difference exposes the type II FAS enzymes as potential
targets for antimicrobial drugs that have little to no side
effects in the human host. A number of inhibitors of type
II FAS enzymes have been discovered - many of which have anti-bacterial
activity. Extensive biochemical and structural studies have
shed light on how these compounds inhibit their target enzymes,
laying the foundation for the design of inhibitors with increased
potency. Recent work has shown that malaria parasites do not
contain a type I FAS and rely solely on a type II FAS for
the de novo biosynthesis of fatty acids. The malaria
FAS enzymes are therefore an exciting source of new drug targets,
and are being actively exploited by several drug discovery
efforts. Rapid progress has been made, largely due to the
vast body of mechanistic and structural information about
type II FAS enzymes from bacteria and the availability of
inhibitors. Ongoing antimalarial drug discovery projects will
be described in this review as well as background information
about the wellstudied bacterial type II FAS enzymes.
[Back to top]
Rational Inhibitor Design and Iterative Screening in the Identification
of Selective Plasmodial Cyclin Dependent Kinase Inhibitors
Susan M. Keenan, Jeanne A. Geyer, William J.
Welsh, Sean T. Prigge and Norman C. Waters
[Full text
article]
New chemical classes of compounds must be introduced into
the malaria drug development pipeline in an effort to develop
new chemotherapy options for the fight against malaria. In
this review we describe an iterative approach designed to
identify potent inhibitors of a kinase family that collectively
functions as key regulators of the cell cycle. Cyclin-dependent
protein kinases (CDKs) are attractive drug targets in numerous
diseases and, most recently, they have become the focus of
rational drug design programs for the development of new antimalarial
agents. Our approach uses experimental and virtual screening
methodologies to identify and refine chemical inhibitors and
increase the success rate of discovering potent and selective
inhibitors. The active pockets of the plasmodial CDKs are
unique in terms of size, shape and amino acid composition
compared with those of the mammalian orthologues. These differences
exemplified through the use of screening assays, molecular
modeling, and crystallography can be exploited for inhibitor
design. To date, several classes of compounds including quinolines
and oxindoles have been identified as selective inhibitors
of the plasmodial CDK7 homologue, Pfmrk. From these initial
studies and through the iterative rational drug design process,
more potent, selective, and most importantly, chemically unique
compound classes have been identified as effective inhibitors
of the plasmodial CDKs and the malarial parasite.
[Back to top]
1,4-Bis(3-Aminopropyl)Piperazine Libraries: From the Discovery
of Classical Chloroquine-Like Antimalarials to the Identification
of New Targets
Rebecca Deprez-Poulain and Patricia Melnyk
[Full text
article]
The purpose of this review is to provide an update on our
work based on the 1,4-bis(3-aminopropyl)piperazine skeleton
and how it allowed our group to validate a new target.
After a brief introduction where we will relate the way this
substructure was introduced in our 4-aminoquinolinyl derivatives,
we will present first the different libraries synthesized
around this moiety: (1) libraries of sulfonamides, amides
and amines derived from 4-aminoquinolines and, (2) libraries
where the 4-aminoquinoline nucleus is replaced. High throughput
evaluation of biological activity and physicochemical parameters
will be presented. The evaluation of the anti-malarial activity
of the compounds will be discussed in the light of a chloroquine-like
mechanism (accumulation in the acidic food vacuole and inhibition
of β-hematin
formation).
In a second part we will present active 1,4-bis(3-aminopropyl)piperazine
as tools for identification and/or validation of new antimalarial
targets. Fluorescence assays on some derivatives show that
they are surprisingly localized outside the food vacuole,
suggesting the existence of other target(s). Secondly, we
will present a library of 1,4-bis(3-aminopropyl)piperazine
as inhibitors of the cytosolic aminopeptidase Pfa-M1, a new
potential target for antimalarials.
[Back to top]
Dual Molecules as New Antimalarials
Xavier J. Salom-Roig, Abdallah Hamze, Michele
Calas and Henri J. Vial
[Full text
article]
A new antimalarial pharmacological approach based on inhibition
of the plasmodial phospholipid metabolism has been developed.
The drugs mimic choline structure and inhibit de novo
phosphatidylcholine biosynthesis. Three generations of compounds
were rationally designed. Bisquaternary ammonium salts showed
powerful antimalarial activity, with IC50 in the
nanomolar range. To remedy their low per os absorption,
bioisosteric analogues (bis-amidines) were designed and exhibited
similar powerful activities. Finally, the third generation
compounds are bis-thiazolium salts and their non-ionic precursors:
prodrugs, which in vivo can lead to thiazolium drugs
after enzymatic transformation.
The compounds are equally effective against multiresistant
Plasmodium falciparum malaria. These molecules exert
a very rapid cytotoxic effect against malarial parasites in
the very low nanomolar range and are active in vivo
against P. vinckei-infected mice, with ED50
lower than 0.2 mg/kg. They are able to cure highly infected
mice and, retain full activity after a single injection. They
also retain full activity against P. falciparum and
P. cynomolgi in primate models with no recrudescence
and at lower doses.
Compounds are accumulated in P. falciparum-infected
erythrocyte, which ensures their potency and specificity.
Recently, we discovered that compounds also interact with
malarial pigment enhancing the antimalarial effect. It is
quite likely that they are dual molecules, exerting their
antimalarial activity via two simultaneous toxic effects on
the intracellular intraerythrocytic parasites. The current
leader compounds are accessible in few steps from commercial
products. These crystalline molecules present a remarkable
biological activity and low toxicity which is promising for
the development of a new antimalarial drug.
[Back to top]
Targeting the Hemozoin Synthesis Pathway for New Antimalarial
Drug Discovery: Technologies for In Vitro β-Hematin
Formation Assay
Babu L. Tekwani and Larry A. Walker
[Full text
article]
Clinical manifestations of malaria primarily result from
proliferation of the parasite within the hosts’ erythrocytes.
During this process, hemoglobin is utilized as the predominant
source of nutrition. The malaria parasite digests hemoglobin
within the digestive vacuole through a sequential metabolic
process involving multiple proteases. Massive degradation
of hemoglobin generates large amount of toxic heme. Malaria
parasite, however, has evolved a distinct mechanism for detoxification
of heme through its conversion into an insoluble crystalline
pigment, known as hemozoin. Hemozoin is identical to β-hematin,
which is constituted of cyclic heme dimers arranged in an
ordered crystalline structure through intermolecular hydrogen
bonding. The exact mechanism of biogenesis of hemozoin in
malaria is still obscure and is the subject of intense debate.
Hemozoin synthesis is an indispensable process for the parasite
and is the target for action of several known antimalarials.
The pathway has therefore attracted significant interest for
new antimalarial drug discovery research. Formation of β-hematin
may be achieved in vitro under specific chemical
and physiochemical conditions through a biocrystallization
process. Based on these methods several experimental approaches
have been described for the assay of formation of β-hematin
in vitro and screening of compounds as inhibitors
of hemozoin synthesis. These assays are primarily based on
differential solubility and spectral characteristics of monomeric
heme and β-hematin.
Different factors viz., the malaria parasite lysate,
lipids extracts, preformed b-hematin, malarial histidine rich
protein II and some unsaturated lipids have been employed
for promoting β-hematin
formation in these assays. The assays based on spectrophotometric
quantification of β-hematin
or incorporation of 14C-heme yield reproducible results and
have been applied to high throughput screening. Several novel
antimalarial pharmacophores have been discovered through these
assays.
[Back to top]
The New Permeability Pathways: Targets and Selective
Routes for the Development of New Antimalarial Agents
Henry M. Staines, J. Clive Ellory and Kelly Chibale
[Full text
article]
The malaria parasite, Plasmodium falciparum, spends
part of its complex life cycle within the red blood cells
of a human host. During this time, the parasite alters the
permeability of the red blood cell’s plasma membrane
to allow the uptake of nutrients, the removal of “waste”
and volume and ion regulation of the infected cell. The increased
permeability is due to the induction of new permeability pathways
(NPP), which are obvious chemotherapeutic antimalarial targets
and/or selective routes for drugs, which target the internal
parasite. This review covers our present understanding of
the NPP, the methods used to screen for putative inhibitors
of the NPP, the current repertoire of NPP inhibitors and the
problems that need to be addressed to realise the potential
of the NPP as antimalarial targets. In addition, the review
will cover the use of the NPP as specific drug delivery routes.
[Back to top]
The Role of In Vitro ADME Assays in Antimalarial
Drug Discovery and Development
Todd W. Shearer, Kirsten S. Smith, Damaris Diaz,
Constance Asher and Julio Ramirez
[Full text
article]
The high level of attrition of drugs in clinical development
has led pharmaceutical companies to increase the efficiency
of their lead identification and development through techniques
such as combinatorial chemistry and high-throughput (HTP)
screening. Since the major reasons for clinical drug candidate
failure other than efficacy are pharmacokinetics and toxicity,
attention has been focused on assessing properties such as
metabolic stability, drug-drug interactions (DDI), and absorption
earlier in the drug discovery process. Animal studies are
simply too labor-intensive and expensive to use for evaluating
every hit, so it has been necessary to develop and implement
higher throughput in vitro ADME screens to manage
the large number of compounds of interest.
The antimalarial drug development program at the Walter Reed
Army Institute of Research, Division of Experimental Therapeutics
(WRAIR/ET) has adopted this paradigm in its search for a long-term
prophylactic for the prevention of malaria. The overarching
goal of this program is to develop new, long half-life, orally
bioavailable compounds with potent intrinsic activity against
liver- and blood-stage parasites. From the WRAIR HTP antimalarial
screen, numerous compounds are regularly identified with potent
activity. These hits are now immediately evaluated using a
panel of in vitro ADME screens to identify and predict
compounds that will meet our specific treatment criteria.
In this review, the WRAIR ADME screening program for antimalarial
drugs is described as well as how we have implemented it to
predict the ADME properties of small molecule for the identification
of promising drug candidates.
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