Combinatorial Chemistry & High Throughput Screening - Volume 8, Issue 1, 2005

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.

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.

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.

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.

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.

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.

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 β-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.

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.

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.

Norman C. Waters obtained his Ph.D. in 1996 from Drexel University (formerly Hahnemann University) working under the supervision of Professor Lawrence W. Bergman in the field of signal transduction and metabolic regulation in the budding yeast Saccharomyces cerevisiae. From 1997-2002, he served as Chief of Anti-Parasite Assay Development in the Division of Experimental Therapeutics at the Walter Reed Army Institute of Research and served as a member of the U.S. Army Antimalarial Drug Discovery Product Development Team. He held the position of Chief of Malaria Drug Discovery and Surveillance at the United States Army Research Unit-Kenya from 2002-2003. He currently serves as Chief of Malaria Drug Target Development in the Division of Experimental Therapeutics at the Walter Reed Army Institute of Research, in addition to technical advisor to the U.S. Army malaria drug discovery efforts in Africa. His interests include: Malaria drug discovery, cell cycle control, cyclin dependent protein kinases, structure based drug design, drug target assay development and validation, signal transduction.