Current Drug Targets - Volume 8, Issue 1, 2007

This theme issue of CDT brings together a number of leading experts in the field of drug (target) discovery against human apicomplexan parasites and highlights major current concepts in this field. Although parasites of the phylum Apicomplexa (single-celled intracellular protozoa) are less well appreciated in public to cause potentially deadly and widespread infectious diseases (like AIDS or influenza), the recently published World malaria report draws a different picture [1]. It states that currently 3.2 billion people are affected by malaria worldwide, killing at least one million people a year, mostly children. Malaria is caused by several species of the apicomplexan parasite Plasmodium able to infect humans, with P. falciparum the most widespread but also the most dangerous species. The fight against malaria has been a top priority of the World Health Organization (WHO) already since its creation in 1948 [2]. In the 1950's there was strong hope that malaria could be eventually eradicated through the widespread use of chloroquine to kill the parasite and DTT to do the same with the Anopheles mosquitos (responsible for malaria transmission). Some malariologists even expressed their concerns that they might have nothing to do in the near future [3]. Now, more than 50 years later, there is no worry that scientists around the world working on malaria or other diseases caused by apicomplexan parasites would be without a job anytime soon. In 107 countries or territories individuals are at risk to contract the disease. In the last 25 years, the burden of malaria has mainly risen because of the general deterioration of primary health services in developing countries, the interruption of eradication measures or due to insecticide-resistant mosquitoes [1]. Moreover, HIV infection also increases the incidence of severe malaria in adults where both diseases are endemic [4]. However, the most important factor responsible for the current malaria situation is the ongoing development of drug resistance in the parasite population [5, 6]. The consequences are that cheap and effective drugs like chloroquine no longer work in most parts of the world. This is a vicious circle since more infected individuals lead to higher transmission rates. All this led to the launch of the Roll Back Malaria (RBM) initiative by WHO, the World Bank, UNICEF and the United Nations Development Program (UNDP) in 1998, with the aim to reduce the global malaria burden by halve by 2010. The time seems right to achieve this goal, since all three genomes of the players involved (Homo sapiens, Plasmodium and Anopheles) are known, and public awareness of the problem seems to have risen, positively affecting also private funding for research on malaria [7]. In addition, combination therapy with drugs having different targets is now a promising approach to treat malaria in areas with widespread resistance to monotherapy [6, 8]. The artemisinin-based combinations are the most promising ones. However, they are very expensive which almost prohibits their widespread use in endemic areas until the plant-derived compounds can be obtained in large amounts and at reasonable prices from precursor molecules [9]. This special issue of CDT is not only about Plasmodium and malaria, however. A number of other Apicomplexa are also important pathogens of humans and livestock. Toxoplasma gondii (causative agent of toxoplasmosis) and Cryptosporidium parvum (causative agent of cryptosporidiosis) are opportunistic pathogens of immunocompromised patients, especially with AIDS, and can lead to either fatal encephalomyelitis (T. gondii) or severe and chronic life-threatening gastroenteritis (C. parvum) if not treated [10, 11]. In addition, T. gondii is also a major cause of congenital infection leading to severe clinical symptoms. Although an acute infection with T. gondii in an immunocompetent person is usually mild and can be controlled by effective drugs if necessary, no proven therapy exists which would be able to eradicate the persistent form of the parasite. Therefore, all currently infected individuals (an estimated 30% worldwide) are at risk should they ever become immune suppressed in the future. In the case of C. parvum no satisfactory medication exists to date, although recent data suggest that highly active antiretroviral therapy (HAART) in HIV-infected individuals also leads to substantially reduced prevalence rates of C. parvum [12]. These and other data might give hints to novel therapies and drug targets [13, 14]. Again, the genomes of both organisms are now known.....

In Plasmodium falciparum the biosynthesis of isoprenoids is achieved by the mevalonate-independent 1-deoxy-D-xylulose 5- phosphate (DOXP) pathway. The enzymes of the DOXP pathway are localised inside the plastid-like organelle (apicoplast). Fosmidomycin inhibits DOXP reductoisomerase, the second enzyme of this pathway. The antimalarial activity of fosmidomycin was established in vitro and in a rodent malaria model. Fosmidomycin alone or in combination with clindamycin was evaluated for the treatment of acute uncomplicated P. falciparum malaria in early phase II studies. Fosmidomycin monotherapy led to a fast parasite and fever clearance but was inefficient in radical elimination of the parasites. With the fosmidomycin-clindamycin combinations the cure ratio on day 28 was 100 % (10/10) with treatment durations of 5 and 4 days. The cure ratio was 90 % (9/10) with treatment duration of 3 days.

Apicomplexan parasitic diseases impose devastating impacts on much of the world's population. The increasing prevalence of drug resistant parasites and the growing number of immuno-compromised individuals are exacerbating the problem to the point that the need for novel, inexpensive drugs is greater now than ever. Discovery of a prokaryotic, Type II fatty acid synthesis (FAS) pathway associated with the plastid-like organelle (apicoplast) of Plasmodium and Toxoplasma has provided a wealth of novel drug targets. Since this pathway is both essential and fundamentally different from the cytosolic Type I pathway of the human host, apicoplast FAS has tremendous potential for the development of parasite-specific inhibitors. Many components of this pathway are already the target for existing antibiotics and herbicides, which should significantly reduce the time and cost of drug development. Continuing interest - both in the pharmaceutical and herbicide industries - in fatty acid synthesis inhibitors proffers an ongoing stream of potential new anti-parasitic compounds. It has now emerged that not all apicomplexan parasites have retained the Type II fatty acid biosynthesis pathway. No fatty acid biosynthesis enzymes are encoded in the genome of Theileria annulata or T. parva, suggesting that fatty acid synthesis is lacking in these parasites. The human intestinal parasite Cryptosporidium parvum appears to have lost the apicoplast entirely; instead relying on an unusual cytosolic Type I FAS. Nevertheless, newly developed anti-cancer and anti-obesity drugs targeting human Type I FAS may yet prove efficacious against Cryptosporidium and other apicomplexans that rely on this Type I FAS pathway.

Synthesis de novo, acquisition by salvage and interconversion of purines and pyrimidines represent the fundamental requirements for their eventual assembly into nucleic acids as nucleotides and the deployment of their derivatives in other biochemical pathways. A small number of drugs targeted to nucleotide metabolism, by virtue of their effect on folate biosynthesis and recycling, have been successfully used against apicomplexan parasites such as Plasmodium and Toxoplasma for many years, although resistance is now a major problem in the prevention and treatment of malaria. Many targets not involving folate metabolism have also been explored at the experimental level. However, the unravelling of the genome sequences of these eukaryotic unicellular organisms, together with increasingly sophisticated molecular analyses, opens up possibilities of introducing new drugs that could interfere with these processes. This review examines the status of established drugs of this type and the potential for further exploiting the vulnerability of apicomplexan human pathogens to inhibition of this key area of metabolism

In evolutionary terms, mitochondria in apicomplexan parasites appear to be “relicts-in-the-making”: they possess the smallest mitochondrial genomes known, encoding only three proteins, and in one genus, Cryptosporidium, the genome is eliminated altogether. Several features of mitochondrial physiology provide validated or potential targets for antiparasitic drugs. Atovaquone, a broad spectrum antiparasitic drug, selectively inhibits mitochondrial electron transport at the cytochrome bc1 complex and collapses mitochondrial membrane potential. Recent investigations using model systems provide important insights into the mechanism of action for this drug, which may prove valuable for development of other selective inhibitors of mitochondrial electron transport. Although mitochondria do not appear to be a source of ATP during the erythrocytic stages in Plasmodium species, they do serve other critical functions, including the assembly of iron-sulfur clusters and various other biosynthetic processes depending on the species. To serve these metabolic functions, parasites need to maintain the apparatus for mitochondrial genome replication, repair, recombination, transcription, and translation, components of which are encoded in the nucleus and imported into the mitochondrion. Several unusual aspects of the components of this apparatus are coming to light through genome sequence analyses, and could provide potential targets for antiparasitic drug discovery and development.

A recent resurgence in the use of compounds to study essential biological processes raises important questions concerning the link between fundamental research and drug development. This article discusses many of the issues involved, in the context of host cell invasion and egress by parasites of the Phylum Apicomplexa. In addition, an overview of the key steps in invasion and egress is provided with a particular emphasis on potential parasite protein drug targets.

The proliferation of the intraerythrocytic malaria parasite is dependent on the uptake from the blood plasma, and from the cytoplasm of the host cell, of a range of essential nutrients. These compounds are taken up into the parasitised cell via a combination of constitutively active endogenous host cell transporters and new parasite-induced permeability pathways. On entering the infected cell they are taken up by the intracellular parasite, across the parasitophorous vacuole and parasite plasma membranes, via a combination of channels and transporters, and/or via endocytosis. Once inside the parasite, nutrients are typically phosphorylated and thereby effectively trapped within the cell. The intraerythrocytic parasite has a range of subcellular membrane-bound organelles, each endowed with their own complement of transport proteins which mediate the uptake and efflux of metabolic substrates and byproducts. Proteins that mediate the uptake, intracellular trafficking and metabolism of essential nutrients in the Plasmodium-infected erythrocyte are potential antimalarial drug targets. Here we consider the nature of the pathways involved, focusing in particular on those that mediate the uptake of three important nutrients: glucose, the key energy-substrate for the parasite; pantothenate (vitamin B5), the precursor of the important enzyme cofactor, coenzyme A; and choline, the precursor of the phospholipid phosphatidylcholine.

G-protein coupled receptors (GPCR) are involved in a large variety of physiological and pathophysiological processes. Their fundamental role is highlighted by the fact that of the around 500 currently marketed drugs, more than 30% are GPCR modulators. GPCR agonist and antagonist drugs have therapeutic benefit across a broad spectrum of diseases, including pain (opioid receptor agonists), asthma ( β2-adrenoceptor agonists), peptic ulcers (histamine H2 receptor antagonists), migraine (serotonin 5-HT1B/1D agonists), hypertension (angiotensin AT2 receptor antagonists), schizophrenia (serotonin 5-HT2 receptor agonists and dopamine receptor antagonists), rhinitis or allergy (histamine H1 receptor and chemokine receptor antagonists), etc. Besides, no single class of proteins ranks higher than GPCRs in terms of new drug discovery potential. It has been estimated that of the around 400 GPCRs considered to be potential drug targets, only ∼30 are targeted by currently marketed drugs. The natural ligand has been identified for a further 210 receptors, which leaves around 160 orphan receptors with no known ligand or function. Without any doubt, therapeutic intervention at these novel receptors will have major benefit in a wide range of human diseases. The matter has been made more complicated by the fact that several GPCR ligands do not interact at the natural ligand binding site, rather such compounds interact elsewhere on the receptor to modulate its activity (allosteric sites). Thus, there may be many as yet uncharacterized drug binding sites within the GPCR that could be exploited for therapeutic intervention. In addition, the recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. GPCRs can be classified into three major families according to sequence homology. Family A is the largest subgroup and includes catecholamine, neuropeptide, chemokine, glycoprotein, lipid and nucleotide receptors. Family B contains receptors for a large number of peptides such as calcitonin gene-related peptide (CGRP) and calcitonin. Family C contains the metabotropic glutamate receptors (mGluRs), γ-amino butyric acid (GABAB) receptors and the calcium-sensing receptor (CaR). Apparently, the large super-family of GPCRs is correlated to a wide range of structurally diverse, heterogeneous ligands. For this reason, a systematic review of the topic is of little utility. Rather, this issue of Current Drug Targets deals with some selected aspects of the current status and future directions of GPCRs investigation, with a particular emphasis on their potential impact on medicinal chemistry. In their review, P. M. Sexton and co. exhaustively discuss the recently de-orphanised relaxin receptors and related ligands. Relaxins (H1, H2 and H3), belong to a peptide family which comprises also insulin, insulin-like peptides (INSL3-6), and insulin-like growth factors (IGF I-II). The relaxin/INSL receptor system is a promising candidates for treatments of problems associated with pregnancy, but also as contraceptive agents, for the treatment of fibrosis, cardiac failure and asthma. The review of P. A. Keller and co. addresses the strategies and the obstacles that have arisen in the search of Corticotrophin Releasing Hormone-based agonists and antagonists for the treatment of anxiety and depression, with additional therapeutic targets including Alzheimer's, pain and the prevention of premature birth. The pharmacology of Nociceptin/Orphanin FQ peptide receptors has been the subject of a couple of articles. In the first one, L.- C. Chiou and co. describe the different kinds of NOP receptor agonists and antagonists, and their possible clinical indications: agonists might be beneficial in the treatment of pain, anxiety, stress-induced anorexia, cough, neurogenic bladder, edema, drug dependence, etc. while antagonists might be of help in the management of pain, depression, dementia and Parkinsonism. In the second one, S. Spampinato and co. discuss the efficacy of NOP receptor agonists to induce receptor endocytosis. Prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways. In the following review, F. Nyberg and M. Hallberg investigate the fate of neuropeptides after receptor stimulation. There is increasing evidence that, besides to peptide inactivation by enzymatic degradation, in many cases the active neuropeptides are enzymatically converted to products that modulate the action of their parent compounds. The contribution of D.R. Herr and J. Chun concerns of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), two lysophospholipids that mediate a diverse range of biological processes......

Relaxin was discovered more than 75 years prior to the identification of the receptors that mediate its actions. There has been a slow emergence in understanding the role of relaxin, with it being denoted initially as a hormone of pregnancy due to its observed effects to relax pubic ligaments and soften the cervix of guinea pigs to facilitate parturition. However, many other physiological roles have been identified for relaxin, including cardiovascular and neuropeptide functions and an ability to induce the matrix metalloproteinases, so it is clear that relaxin is not exclusively a hormone of pregnancy but has a much wider role in vivo. The recent de-orphanisation of four receptors LGR7, LGR8, GPCR135 (SALPR) and GPCR142 (GPR100) that respond to and bind at least one of the three forms of relaxin identified to date, allows dissection of this system to determine the precise role of each receptor and enable the identification of new targets for treatment of numerous disease states. Relaxin has the potential to be useful for the treatment of scleroderma, fibrosis, in orthodontics and to facilitate embryo implantation in humans. Relaxin antagonists may act as contraceptives or prevent the development of breast cancer metastases. Recent research has added considerable knowledge to the signalling pathways activated by relaxin, which will aid our understanding of how relaxin produces its effects. The focus of this review is to bring together recent developments in the relaxin receptor field and to highlight their potential as drug targets.

Corticotrophin Releasing Hormone (CRH) is a primary hormone in the fight or flight response targeting a membrane bound Gprotein coupled receptor (GPCR). Many people worldwide stand to benefit by the development of CRH agonists and antagonists for the treatment of anxiety and depression, with additional therapeutic targets including Alzheimer’s, pain and the prevention of premature birth: so why the delay in development? In this review, we will discuss not only CRH, related proteins, receptors and ligands, but some of the obstacles that have arisen, as well as strategies being pursued to overcome these problems in the pursuit of this GPCR targeted therapeutic. Several key proteins influence the complex and intrinsic regulation of CRH, including its receptors (CRHR), of which 3 types have been categorised, CRHR1, CRHR2, CRHR3, each containing active and inactive splice variants. Additionally, the CRH binding protein (CRHBP) is believed to moderate the effects of CRH at the receptor, whether it is as a molecular mop, or a delivery vessel, or both, is still being investigated. Homology based receptor modelling is a technique that has only recently become available with the crystallisation of bovine rhodopsin (a GPCR), [1] and the application of this technique to the CRH receptors is still in the early stages of development. Therefore, the medicinal chemist has previously had to rely on ligand-based strategies, specifically, the development of pharmacophores. Thus, an extensive number of both CRH peptide analogues and small ligands that show nanomolar antagonism have been developed with SAR libraries being integral to the iterative drug design process.

The advance of functional genomics revealed the superfamily of G-protein coupled receptors (GPCRs). Hundreds of GPCRs have been cloned but many of them are orphan GPCRs with unidentified ligands. The first identified orphan GPCR is the opioid receptor like orphan receptor, ORL1. It was cloned in 1994 during the identification of opioid receptor subtypes and was de-orphanized in 1995 by the discovery of its endogenous ligand, nociceptin or orphanin FQ (N/OFQ). This receptor was renamed as N/OFQ peptide (NOP) receptor. Several selective ligands acting at NOP receptors or other anti-N/OFQ agents have been reported. These include N/OFQ-derived peptides acting as agonists (cyclo[Cys10,Cys14]N/OFQ, [Arg14, Lys15]N/OFQ, [pX]Phe4N/OFQ(1-13)-NH2, UFP-102, [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2) or antagonists (Phe1ψ (CH2-NH)Gly2]N/OFQ(1-13)-NH2, [Nphe1]N/OFQ(1-13)-NH2, UFP-101, [Nphe1, (pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2), hexapeptides, other peptide derivatives (peptide III-BTD, ZP-120, OS-461, OS-462, OS- 500), non-peptide agonists (NNC 63-0532, Ro 64-6198, (+)-5a compound, W-212393, 3-(4-piperidinyl)indoles, 3-(4-piperidinyl) pyrrolo[2,3-b]pyridines) and antagonists (TRK-820, J-113397, JTC-801, octahydrobenzimidazol-2-ones, 2-(1,2,4-oxadiazol-5-yl)-1 Hindole, N-benzyl-D-prolines, SB-612111), biostable RNA Spiegelmers specific against N/OFQ, and a functional antagonist, nocistatin. Buprenorphine and naloxone benzoylhydrazone are two opioid receptor ligands showing high affinity for NOP receptors. NOP receptor agonists might be beneficial in the treatment of pain, anxiety, stress-induced anorexia, cough, neurogenic bladder, edema, drug dependence, and, less promising, in cerebral ischemia and epilepsy, while antagonists might be of help in the management of pain, depression, dementia and Parkinsonism. N/OFQ is also involved in cardiovascular, gastrointestinal and immune regulation. Altered plasma levels of N/OFQ have been reported in patients with various pain states, depression and liver diseases. This review summarizes the pharmacological characteristics of, and studies with, the available NOP receptor ligands and their possible clinical implications.

In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHOK1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH2, NC(1-13)-NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH2, NC(1-13)- NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 (5 μM) ≈20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The noninternalizing agonists [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 (100 μM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways.

Previous and current research has revealed that most neuropeptides induce their actions on cellular systems through specific receptors located on the cell surface. These receptors are known as G-protein coupled receptors, which exert their effects through interaction with ion channels or enzymes located within the cell membrane. Following receptor stimulation and exerting their effects the peptides are inactivated by enzymatic degradation. However, in many cases the active neuropeptides are enzymatically converted to products with retained bioactivity. These bioactive fragments may mimic but also counteract the action of the parent peptide. Thus, the released fragment may serve as a modulator of the response of the original compound. This phenomenon has been found to occur in a number of peptide systems, including the opioid peptides, tachykinins, as well as peptides belonging to the renin-angiotensin system, such as angiotensin II. In some cases the conversion product interacts with the same receptor as the native compound but sometimes it appears that the released fragment interacts with receptors or binding sites distinct from those of the original peptide. This review is focused on peptide fragments released from opioid related peptides, substance P and angiotensin II, that have been shown to modulate the action of their parent compounds.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are two well-studied lysophospholipids that are known to be important regulators of cellular events. Their actions are mediated by activating a family of G-protein coupled receptors present in many cell types and tissues. These receptors have diverse biological roles owing to the heterogeneity of their signal transduction pathways. Many of these receptors are expressed in subsets of cells in the developing and mature mammalian nervous system and are thought to have important functions in its formation and maintenance. They are also widely expressed within other organ systems such as the immune system. Growing interest in the field has stimulated the development of a number of molecules that act as agonists or antagonists to LPA and S1P receptors. These molecules may lead to the development of new therapeutic compounds. Indeed, one such compound (FTY720) is currently in clinical trials for use in preventing transplant rejection and treating multiple sclerosis. The purpose of this manuscript is to: 1) review effects elicited by LPA and S1P on cells and tissues with a particular emphasis on the nervous system, 2) examine possible roles of these lipids in the development of disease, and 3) summarize the existing literature describing their agonists/antagonists.

Family C of G-protein coupled receptors (GPCRs) from humans is constituted by eight metabotropic glutamate (mGlu1-8) receptors, two heterodimeric -aminobutyric acidB (GABAB) receptors, a calcium-sensing receptor (CaR), three taste (T1R) receptors, a promiscuous L- α-amino acid receptor (GPRC6A), and five orphan receptors. Aside from the orphan receptors, the family C GPCRs are characterised by a large amino-terminal domain, which bind the endogenous orthosteric agonists. Recently, a number of allosteric modulators binding to the seven transmembrane domains of the receptors have also been reported. Family C GPCRs regulate a number of important physiological functions and are thus intensively pursued as drug targets. So far, two drugs acting at family C receptors (the GABAB agonist baclofen and the positive allosteric CaR modulator cinacalcet) have been marketed. Cinacalcet is the first allosteric GPCR modulator to enter the market, which demonstrates that the therapeutic principle of allosteric modulation can also be extended to this important drug target class. In this review we outline the structure and function of family C GPCRs with particular focus on the ligand binding sites, and we present the most important pharmacological agents and the therapeutic prospects of the receptors.

Among the many receptor classes of the GPCR family, ORs constitute a privileged drug target for their involvement in pain modulation and in a number of physiological functions and behavioural effects. Endogenous and exogenous opioid agonists have been the subject of intense investigations aiming to develop safe and potent analgesics for clinical practice; however, despite the large number of highly selective opioid agonists so far discovered, there is no convincing alternative to the use of morphine, fentanyls, and their derivatives. Alternative compounds could be very useful for treating pain forms “resistant” to the usual therapeutic agents. The recent discovery of a small number of atypical opioid agonists can furnish promising candidates for the development of alternative analgesic. In particular, a few molecules exist that can bind and activate ORs even deprived of the “minimal pharmacological requisites” generally considered to be necessary. In these cases it appears that receptor activation must be based on different ligand-receptor interaction mechanisms. Taken together, the data discussed in the review suggest that the prevailing assumptions about OR binding need revision. In particular, they strengthen the evidence that ORs can bind ligands via diverse binding modes, and in some cases an electrostatic interaction is not an absolute requirement.

Classical methods for the estimation of antagonist affinity constants were developed under the assumption of one unique state for the receptor. The finding of receptor constitutive activity, which implies that at least two (one active and the other inactive) receptor states coexist at equilibrium, extended the concept of antagonism by distinguishing between neutral antagonists and inverse agonists. To account for the complexity introduced in the concept of antagonism, classical Schild and Cheng-Prusoff methods have been revisited within the two-state model of agonism. The resulting equations match the classical expressions for neutral antagonists but not for inverse agonists. It is suggested a revision of current routine procedures for antagonist affinity estimation.