Current Topics in Medicinal Chemistry - Volume 7, Issue 4, 2007

The rapid advances of molecular biology, neuropharmacology, anatomy, gene cloning, and recombinant DNA techniques have unravelled our understanding of a large number of receptors. An increasing number of secondary and tertiary structures of these receptors are being disclosed. Further, an increasing number of receptors have been reported recently to dimerize or oligomerize, which opens a new vistas to receptor researchers. The biology, pharmacology, function and utility of such phenomenon, as well as how receptor dimerization/ oligomerization can guide drug design and discovery are being extensively investigated. In this issue of Current Topics in Medicinal Chemistry, we highlight several aspects of the receptor dimerization and bivalent ligands of the most developed receptors by collecting comprehensive reviews from several experts working in this emerging field. In the first article, Zhang and Kan discussed the most recent advances in the receptor dimerization phenomenon. Several key elements regarding this process are postulated and discussed. The utility of this common feature of receptors to guide drug design and development are confirmed, and the critical considerations leading to the successful discovery of bivalent drugs are presented. Decker and Lehmann reviewed bivalent ligands developed for dopamine and serotonin receptors, as well as their respective transporters reported within the last two decades. They pointed out that increased potency, selectivity and CNS penetration for 5-HT1B/1D receptor agonists could be achieved with the bivalent ligands. Bivalent dopamine receptor agonists and antagonists can exhibit selectivity profiles very different from their monomeric analogues without loss in potency. Nowak presented the progress of bivalent ligands for probing the membrane targeting C1 subdomains of protein kinase C (PKC), a family of ubiquitously expressed signal transducing proteins. Novel and conventional subfamilies of PKC have two C1 domains, C1A and C1B. Bivalent PKC ligands are designed to activate simultaneously both C1 domains. Many bivalent ligands displayed 1-2 orders of magnitude higher potency than their monovalent congeners with the “dimeric” ligands containing 10-14 carbon spacers being the most effective. Peng and Neumeyer presented a comprehensive review of the most developed kappa(κ) opioid receptor bivalent ligands. κ Agonists and antagonist ligands such as norBNI and BNI have been used as tools to elucidate the κ receptor characteristics. Bivalent ligands may also be effective analgesics although none have this far been used clinically. Structure-activity relationships and molecular modeling has led to the development of a more potent and selective κ antagonist (GNTI). In addition, novel hetero-bivalent ligands with high mixed κ/μ or mixed κ/δ affinity and intriguing pharmacological properties are also discussed. It is pointed out that such bivalent ligands have great potential as novel analgesics with fewer adverse side effects, and as alternative treatment for drug abuse. Haviv, Wong, Silman and Sussman commented on the bivalent ligands derived from Huperzine A as an acetylcholinesterase (AchE) inhibitors. The naturally occurring alkaloid Huperzine A (HupA) has been used for centuries as a Chinese folk medicine from its source plant Huperzia Serrata. Its pharmacological profile has now led to its use as a promising drug for the treatment of Alzheimer's disease. Biochemical and crystallographic studies of AChE revealed two potential binding sites in the active-site of AChE, initiating the development of Huperzine A-based bivalent ligands. Extensive studies have been conducted by these authors as well as by others, and are summarized in this manuscript. The advantages and disadvantages of such a bivalent ligand approach are also presented.

Accumulating evidence has confirmed the existence and functional significance of GPCR and other types of receptor homodimers and heterodimers. However, many aspects of the biology and pharmacology of the dimerization process remain unclear. Several crystal structures of the dimerized proteins or protein-ligand complex have provided useful insights to the understanding of such process. As a common phenomenon of most receptors, homodimerization and heterodimerization have added a new dimension to characterize the receptor different from the traditional way, and open a new avenue to rational drug design and discovery.

This review deals with the literature (1982-2006) concerning bivalent ligands for dopamine (D) and serotonin (5-HT) receptors, as well as for their respective transporters. The design, synthesis, and pharmacological evaluation of bivalent agonists and antagonists for dopamine and serotonin receptors have been successfully pursued. Increased potencies for 5-HT1B/1D receptor agonists were achieved as well as improved selectivities. At these receptors, selectivity seems to depend strongly on spacer length, whereas the improved affinities seem to be based on the presence of two pharmacophores within one molecule. Intrinsic activities and pharmacokinetic properties may differ from those of the respective monovalent ligands. Additionally, improved central nervous system penetration was achieved. Bivalent dopamine receptor agonists and antagonists can exhibit selectivity profiles different from their monomeric analogues with no loss in potency. For dopamine antagonists, affinities depend strongly on spacer length. For agonistic dimers different pharmacokinetic properties were observed. Bivalency was also applied to inhibitors of monoamine re-uptake transporters. Selectivity profiles and affinities depend strongly on the length of the alkylene-spacer: For some dimeric inhibitors the norepinephrine transporter (NET) and the dopamine transporter (DAT) affinities changed gradually, but for the serotonin transporter (SERT) a pentamethylene spacer showed the highest potency. Because the bivalent ligand approach has just begun to be applied to these versatile, therapeutically important targets, many advances in affinity enhancement, as well as the achievement of novel selectivity profiles and improved pharmacokinetics can be expected.

Protein kinase C is a family of ubiquitously expressed signal transducing proteins. The hallmark for PKC activation is its translocation to membranes following generation of lipid second messengers. This process is mediated by C1 and C2 membrane-targeting modules, whose engagement on membranes provides energy for a conformational change crucial to PKC activity. Novel and conventional subfamilies of PKC have two C1 domains, namely C1A and C1B, each of which contain a binding pocket for a messenger. Several studies addressed the issue of simultaneous activation of both C1 domains by specifically designed bivalent activators based on phorbol esters, benzolactam and other PKC ligands. Many bivalent ligands displayed 1-2 orders of magnitude higher potency then their monovalent congeners. Most effective were the “dimeric” ligands linked with 10-14 carbon spacers. Lower than predicted potency and lack of marked isoform selectivity indicate that those compounds do not activate both C1 domains at the same time, or that process is unfavored due to steric or conformational reasons. However, high binding affinity for some of them provides hope that related PKC activators that are isoform selective can be developed. As to the nature of the linkers: flexible and lipophilic oligomethylene chains proved superior over flexible and hydrophilic oligoethylene glycol or rigid and lipophilic benzene in recruiting PKC to the membranes.

Bivalent ligands of κ opioid agonists and antagonists, such as norBNI and BNI, are used as tools to elucidate the κ receptor characteristics. Bivalent ligands may also be effective analgesics although none have this far been used clinically. Structure-activity relationships (SAR) and molecular modeling led to the development of a more potent and selective kappa antagonist (5'-guanidinylnaltrindole, GNTI). Novel homo and hetero bivalent ligands with high mixed κ/μ or mixed κ/δ affinity and intriguing pharmacological properties may eventually lead to useful analgesics with fewer adverse side effects. Bivalent ligands were also developed that could act as probes of the receptor-oligomerzation and organization phenomena. A structurally unique κ antagonist (JDTic) provides an additional tool to characterize κ opioid receptor.

The naturally occurring alkaloid Huperzine A (HupA) is an acetylcholinesterase (AChE) inhibitor that has been used for centuries as a Chinese folk medicine in the context of its source plant Huperzia Serrata. The potency and relative safety of HupA rendered it a promising drug for the ameliorative treatment of Alzheimer's disease (AD) vis-a-vis the “cholinergic hypothesis” that attributes the cognitive decrements associated with AD to acetylcholine deficiency in the brain. However, recent evidence supports a neuroprotective role for HupA, suggesting that it could act as more than a mere palliative. Biochemical and crystallographic studies of AChE revealed two potential binding sites in the active-site gorge of AChE, one of which, the “peripheral anionic site” at the mouth of the gorge, was implicated in promoting aggregation of the beta amyloid (Aβ) peptide responsible for the neurodegenerative process in AD. This feature of AChE facilitated the development of dual-site binding HupA-based bivalent ligands, in hopes of concomitantly increasing AChE inhibition potency by utilizing the “chelate effect”, and protecting neurons from Aβ toxicity. Crystal structures of AChE allowed detailed modeling and docking studies that were instrumental in enhancing the understanding of underlying principles of bivalent inhibitor-enzyme dynamics. This monograph reviews two categories of HupA-based bivalent ligands, in which HupA and HupA fragments serve as building blocks, with a focus on the recently solved crystallographic structures of Torpedo californica AChE in complex with such bifunctional agents. The advantages and drawbacks of such structured-based drug design, as well as species differences, are highlighted and discussed.

In this issue of Current Topics in Medicinal Chemistry, four review articles have been compiled to exemplify the state-ofthe- art in the design and application of phosphodiesterase inhibitors. This is not a new topic; for fifty years chemists have been called upon to provide novel compounds that inhibit this family of enzymes and it has been these compounds, in league with advances in cellular and molecular biology that have helped unravel the complexities of the cyclic nucleotide second messenger systems. In the last decade however, one advance has thrust this research area back to the forefront of contemporary drug design - the successful clinical application of sildenafil (Viagra) as a medication for male erectile dysfunction (MED). The commercial and clinical success of Viagra, a PDE5 inhibitor and its PDE5 inhibiting competitors, vardenafil and tadalafil, essentially won the argument that PDE inhibitors could be successful modern drugs, and thus provided the evidential weight to push PDE-based research proposals into fruition in industry and academia alike. Of course, PDE5 is but one isoform of this enzyme family, which continues to grow with gene splicing variants emerging across the various pharmacologically and genomically defined family of 11 members including over 60 sub-types. Significant drug discovery efforts have resulted in a number of current clinical candidates against PDE4-related diseases, and significant research efforts against other isoforms. Overall though, the capacity to identify PDE isoforms currently outstrips our ability to characterise their roles in physiology or pathology, and the state of knowledge regarding many of the PDE isoforms is superficial or controversial. This brings us to something of a Catch 22 in respect of advance in the PDE field. Typically, medicinal chemistry campaigns are not undertaken without some observation of a therapeutic potential relating to a selected biological target. However, in the case of the majority of PDEs, such therapeutic potential still is some distance short of validation. This is mainly due to the lack of pharmacological inhibitors. It is thus down to the groups with the curiosity and opportunity to look, not just for drugs but for the crucial reagents which will fuel discovery biology in what is a fundamental component of the circuitry of the cell. In deference to the most significant recent technological breakthroughs, the articles show the powerful application of x-ray crystallography as a tool for drug discovery. Hengming Ke, who was the first to describe the crystal structure of the catalytic domain PDE4, together with Huanchen Wang surveys the burgeoning crystal data released since 2000, to explore what that information tell us about the origins of substrate specificity and inhibitor selectivity across PDE classes. Another contribution comes from the laboratories where Viagra was developed at Pfizer in Sandwich, UK. Michael Palmer, Andrew Bell, David Fox and David Brown show the power of high throughput crystallography and screening, in accelerating the hit to lead process for discovery of second generation PDE5 inhibitors. A pharmacological perspective on PDE5 beyond MED is provided by Bing Zhu and Samuel Strada, from University of Sth. Alabama, who have examined the role of that enzyme together with other cGMP-hydrolyzing isoforms in diseases such as cancer with the clinical candidate, Exisulind and its analogues. They also explore the return of PDE5 inhibition to its “first home” in the study of pulmonary hypertension, and describe the discoveries that are validating PDE5 inhibition as a target in this therapeutic area. Finally, Eva Degerman, Vince Manganiello and I have turned our attention to the need for new PDE3 inhibitors as pharmacological tools and potential therapeutics. An area largely neglected, since the 1990's on the back of clinical failures, the advances in molecular biology, structural biology and cell physiology demand revived activity in the area. We attempt to distill the mass of medicinal chemistry from the 1980's and 1990's to discover new opportunities in the field, particularly with respect to PDE3 sub-types, PDE3A and PDE3B. Necessarily, these articles are limited to reviewing a small subset of the range of isoforms of current and future interest. Inhibitors of those less famous isoforms such as PDE2, PDE7 and PDE9 are emerging regularly in the patent and peer-reviewed literature and it seems likely that there maybe a real prospect of having available isoform-selective inhibitors at PDEs 1 - 11, and many that can distinguish between sub-types. Hopefully however, there is something to be retrieved in each review for researchers in the field of PDE inhibition that might inspire a worthwhile experiment. I thank Dr. Allen Reitz for the invitation to prepare this collection; it is humbling to present the offerings of a number of major players in the field of PDE research and I thank them for their willingness to participate and embrace the themes of this issue. Their conceptual and technical approaches are certainly those that will advance research in the PDE field.

Crystal structures of seven phosphodiesterase families (PDE1-5, 7, 9) show a conserved core catalytic domain that contains about 300 amino acids and fourteen α-helices. The catalytic domains of the PDE families 1-4, 7, and 9 have a uniform conformation. However, the H-loop at the active site of PDE5 shows four different conformations upon binding of inhibitors, probably implying a special mechanism for recognition of substrates and inhibitors by PDE5. The active site of all PDE families contains two divalent metal ions: zinc and probably magnesium. The PDE4-AMP and PDE5-GMP structures reveal the conserved interactions of the phosphate groups of the products AMP and GMP, and thus suggest a universal mechanism of nucleophilic attack for all PDE families. The substrate specificity has not been well understood. This review will comment on the early proposal, “glutamine switch”, on basis of the recent biochemical and structural information. The PDE-inhibitor structures have identified a common subpocket for non-selective binding of all inhibitors and potential elements for recognition of familyselective inhibitors. The kinetic analysis on the mutations of PDE7 to PDE4 suggests that the multiple elements must work together to define inhibitor selectivity.

The clinical significance of phosphodiesterase 5 (PDE5) inhibition is increasingly understood following the pioneering work with sildenafil, and the continuing development programmes for both sildenafil and other marketed inhibitors. Since the initial launch of sildenafil for male erectile dysfunction (MED), approval has now been granted for treatment of pulmonary hypertension, whilst ongoing studies have indicated the potential of PDE5 inhibition for the treatment of a range of additional indications including cardioprotection, memory retention and diabetes. Many of these additional indications are best suited to chronic oral dosing and emphasise the need for highly selective inhibitors with extended duration of action. This article will focus on a research programme aimed at the discovery of improved secondgeneration PDE5 inhibitors. Essential features of these new PDE5 inhibitors would be enhanced selectivity across the whole PDE family and pharmacokinetics compatible with once daily dosing. Key elements used in this programme are high throughput screening (HTS), exploitation of co-crystal structural information for bound inhibitor in the PDE5 active site, and employment of parallel chemistry to speed progress. Under the guidance of co-crystal structural information, a non-selective HTS hit with poor physicochemistry was initially modified using parallel chemistry to give a lead compound (3) that established a new PDE5 inhibitor series. Notably, (3) displayed physicochemistry compatible with a long plasma half-life, and wide chemical scope. Subsequent optimisation of (3) using crystal structure information to guide design, led rapidly to highly potent and selective PDE5 inhibitors (47, 50). Continued focus on physical properties through ligand efficiency evaluation and lipophilicity (cLogP), maintained the inherently desirable physicochemistry of the initial lead.

The PDE3 enzymes or “low Km cGMP-inhibited phosphodiesterases” have long been established as important mediators of cellular physiology, and synthetic PDE3 inhibitors have been critical to the delineation of the enzymes' roles. Yet despite decades of progress on the biology of these enzymes, the medicinal chemistry landscape relating to PDE3 inhibitors has remained essentially unchanged since the mid 1990's. Up until then the field was at the cutting edge of drug design; without the tools of molecular and structural biology, molecules of high potency were being achieved using logical pharmacophore models and lead modification. Yet virtually all the impetus went out of this area on the back of failures at the clinic and PDE3 as a therapeutic target largely fell out of favour. A decade later and with the “new” technologies of structural and molecular biology breathing new life into PDE3 research in general, PDE3 inhibitors are sought for target validation in an array of therapeutic applications. In this review, we examine the current state of PDE3 research; firstly we summarize the structural and functional properties of PDE3 enzymes with particular attention to the heterogeneity within this class of enzymes which differ markedly in expression, localisation and means of regulation across various tissue types. It is the structural and functional complexity of the PDE3 enzymes that underpins the re-emergence of PDE3s roles as targets for drug design. We then look at past clinical evaluation of PDE3 inhibitors that occurred without that information and which may have had a significant bearing on the outcome of those drug discovery efforts. Finally we look at current approaches to the design of PDE3 inhibitors which utilize that historic data but also incorporate new inputs from structural biology and combinatorial chemistry.

PDE5 is a key enzyme involved in the regulation of cGMP-specific signaling pathways in normal physiological processes such as smooth muscle contraction and relaxation. For this reason, inhibition of the enzyme can alter those pathophysiological conditions associated with a lowering cGMP level in tissues. For example, selective PDE5 inhibitors, such as sildenafil (Viagra, Pfizer), tadalafil (Cialis, Lilly-ICOS), and vardenafil (Levitra, Bayer), have been successfully used to treat the condition of human erectile dysfunction. More recently, the involvement of this enzyme has been proposed to influence antiproliferation and proapoptotic mechanism in multiple carcinomas. The data supporting this idea is based on increases in PDE5 activities in many carcinomas and the ability of PDE5 inhibitors such as exisulind and its analogs related to anticancer activities. Inhibition of PDE5 that results in sustained increases in [cGMP]i are required to modify the process of apoptosis and mitotic arrest in those carcinoma cells with enhanced PDE5 expressions. Increases in PDE5 are also involved in contributing to the pathological changes in the pulmonary system resulting in hyperproliferative remodeling of both smooth muscle and endothelium in models of pulmonary hypertension. For this reason, the use of PDE5 inhibitors in the treatment of human pulmonary hypertension has met with some success. The differences that we have previously noted in PDE isoenzymes in pulmonary arterial and microvascular endothelial cells may provide a more selective cellular strategy for use of such inhibitor. Additional studies on structure biology of these enzymes should lead to the development of agents with better cellular specificity than currently available drugs. Considering the enormous progress that has been made in the last few years, the future looks promising for agents affecting this enzyme and related systems.

A significant portion of the manuscript of Loiseau, P. M. and Bories C. entitled “Mechanisms of Drug Action and Drug Resistance in Leishmania as Basis for Therapeutic Target Identification and Design of Antileishmanial Modulators” Curr. Top. Med. Chem. 2006, 6, 539-550 was copied verbatim from the paper of M. Ouellette, J. Drummelsmith, and B. Papadopoulou entitled “Leishmaniasis: drugs in the clinic, resistance and new developments” Drug Res. Updates 2004, 7, 257-266. Therefore, Curr. Top. Med. Chem. has formally retracted the citation Loiseau, P. M.; Bories, C. Curr. Top. Med. Chem. 2006, 6, 539-550 from publication.