Action Of Allosteric Modulators Research Articles

Sensing conformational changes in metabotropic glutamate receptors

G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are targets of more than 25% of the medications currently on the market. Metabotropic glutamate receptors (mGluRs), family C GPCRs, modulate synaptic transmission and neuronal excitability throughout the central nervous system and thus are promising targets for the treatment of various neurological and psychiatric disorders (1). Understanding the detailed activation mechanisms of mGluRs is therefore crucial to the development of new therapeutics. Although extensively studied over the last decade, the conformational changes involved in receptor activation are still a matter of debate (2). In PNAS, Doumazane et al. (3) describe how they have developed an innovative FRET-based approach to monitor these conformational changes upon ligand binding. Their assay not only helps to resolve controversy regarding the conformational changes involved in receptor activation but also facilitates our understanding of the mode of action of allosteric modulators. This approach therefore provides a powerful tool for drug screening.

Editorial (Hot Topic: From Allosteric Drugs to Allo-Network Drugs: State of the Art and Trends of Design, Synthesis and Computational Methods)

Allosteric drugs bind to sites which are usually less conserved evolutionarily as compared to orthosteric sites. As such, they can discriminate between closely related proteins, have fewer side effects, and a consequent lower concentration can convey a lesser likelihood of receptor desensitization. However, an allosteric mode of action may also make the results of preclinical and animal experiments less predictive. The sensitivity of the allosteric consequences to the environment further increases the importance of accounting for patient population diversity. Even subtle differences in protein sequence, in cellular metabolic states or in target tissues, can result in different outcomes. This mini-hot-topic issue of CTMC showcases some successes and challenges of allosteric drug development through the examples of seventransmembrane (GPCR), AMPA, NMDA and metabotropic glutamate receptors, as well as the morpheein model of allosterism involved in inherent metabolic errors. Finally, the development of allo-network drugs, which are allosteric drugs acting indirectly on the neighborhood of the pharmacological target in protein-protein interaction or signaling networks, is described.

Attacking MALT1 for ABC-DLBCL therapy

Diffuse large B-cell lymphoma (DLBCL) comprises the largest group of non-Hodgkin lymphoma (NHL). DLBCL can be classified according to their cellular origin and patients with the activated B-cell (ABC) subtype have an inferior prognosis relative to those with the germinal center B-cell (GCB) subtype. Constitutive anti-apoptotic NF-κB signaling is a hallmark of ABC-DLBCL. NF-κB activation is caused by chronic active B-cell receptor (BCR) signaling or constitutive MYD88 signaling. Oncogenic mutations that contribute to pathogenesis of ABC-DLBCL tumors have been identified in critical components of both pathways (Figure ​(Figure1)1) [1]. Figure 1 Oncogenic mechanisms and target directed therapeutic approaches in ABC-DLBCL Target directed approaches for ABC-DLBCL therapy have largely focused on the inhibition of upstream protein kinase [2]. Chronic BCR signaling engages the adaptors CD79A and CD79B in a Syk-dependent mechanism. Syk is constitutively active in many B-cell lymphomas and a clinical phase I/II trial using the Syk inhibitor fostamatinib disodium (FosD, AstraZeneca) shows some response also in DLBCL patients. However, most oncogenic mutations in ABC-DLBCL occur further downstream revealing that Syk may not be an optimal target. Downstream of CD79A/B, Btk and PKCβ bridge proximal BCR signaling events to the CARMA1 (CARD11)-BCL10-MALT1 (CBM) complex. Over 20% of ABC-DLBCL tumors carry oncogenic mutations in CD79A/B. Indeed, the irreversible Btk inhibitor ibrutinib (PCI-32765, Pharamcyclics) and the panPKC inhibitor sotrastaurin (STN) are inhibiting the outgrowth of CD79 mutant ABC-DLBCL in preclinical models [3, 4]. Furthermore, positive clinical responses in a phase II trial were reported in relapsed/refractory DLBCL with the selective PKCβ inhibitor enzastarin (LY317615, Eli Lilly) [5]. However, none of these potential drugs is able to target ABC-DLBCL tumors with lesions downstream of PKCβ or in parallel pathways, such as CARMA1 of MYD88, respectively. Downstream of PKCβ the CBM subunit MALT1 has attracted great attention as a potential therapeutic target [6]. MALT1 serves a dual role in NF-κB signaling in response to antigen receptor stimulation. MALT1 is an adaptor that recruits and activates the IκB kinase (IKK) complex, the gatekeeper of canonical NF-κB. In addition, MALT1 is a paracaspase with a caspase-like proteolytic activity that is required for full NF-κB signaling and survival of ABC-DLBCL cells. MALT1 cleaves and thereby inactivates negative regulators of canonical NF-κB, such as the tumor suppressor A20 and the NF-κB subunit RelB which both counteract pro-survival functions of canonical IKK/NF-κB signaling. Two classes of MALT1 inhibitors have now been identified that effectively and selectively inhibit the growth of ABC- but not GCB-DLBCL in vitro and in vivo [7, 8]. Interestingly, the compounds are inhibiting MALT1 by two very different mechanisms. Fontan et al have identified a structurally new small molecule inhibitor (MI-2) that is covalently modifying catalytic center of MALT1 [7]. MI-2 was tolerated in mice at the effective dose without obvious signs of toxicity. Usually, irreversible inhibitors require optimal pharmacokinetic properties for clinical development, but recent advances for instance on the irreversible Btk inhibitor ibrutinib reveal that a clinical use may be possible. In a parallel study, we have identified the phenothiazines-derivatives (PD) thioridazine, mepazine and promazine as reversible MALT1 inhibitors [8]. PD are not targeting the active site of MALT1, but exhibit a non-competitive, allosteric mode of action. Mepazine, thioridazine and promazine have a long medical history as antipsychotics and sedatives drugs used for the treatment of psychiatric disorders. Well-established toxicokinetics and pharmacokinetics suggest that targeting MALT1 for cancer therapy by this class of compounds may be safe and feasible. Further, medicinal chemistry could be used to generate novel PD that are more potent MALT1 inhibitors, while reducing their neurological effects. Taken together, both studies demonstrate that MALT1 inhibition is a promising strategy for the treatment of ABC-DLBCL. In fact, targeting MALT1 may possess some advantages over the inhibition of upstream protein kinases. MALT1 inhibition also affects survival of CARMA1 mutant ABC-DLBCL. Further, with an occurrence of 29% the MYD88 mutation L265P is the most frequent oncogenic mutation in ABC-DLBCL. 65% of the MYD88-mutant ABC-DLBCL tumors carry additional mutations in CARMA1 or CD79A/B and MALT1 inhibitors are toxic to ABC-DLBCL with aberrant activation of both pathways [7, 8]. Thus, MALT1 inhibition indeed holds great promises for the treatment of the majority of ABC-DLBCL. Research on ABC-DLBCL provides a paradigm for the power of using advanced diagnostic tools (e.g. gene expression profiling and genome sequencing) to classify lymphoma entities and to identify specific oncogenic lesions. In parallel the generation of target directed therapeutics will promote the development of more personalized treatment protocols. Because of the various oncogenic lesions and the possibilities of drug resistances, there cannot be enough ‘smart’ drugs for ABC-DLBCL therapy. In the future, combinatorial treatment protocols may certainly enhance anti-tumor efficacy of the single agents that are currently in preclinical and/or clinical development.

Fragment Screening of GPCRs Using Biophysical Methods: Identification of Ligands of the Adenosine A2A Receptor with Novel Biological Activity

Fragment-based drug discovery (FBDD) has proven a powerful method to develop novel drugs with excellent oral bioavailability against challenging pharmaceutical targets such as protein-protein interaction targets. Very recently the underlying biophysical techniques have begun to be successfully applied to membrane proteins. Here we show that novel, ligand efficient small molecules with a variety of biological activities can be found by screening a small fragment library using thermostabilized (StaR) G protein-coupled receptors (GPCRs) and target immobilized NMR screening (TINS). Detergent-solubilized StaR adenosine A(2A) receptor was immobilized with retention of functionality, and a screen of 531 fragments was performed. Hits from the screen were thoroughly characterized for biochemical activity using the wild-type receptor. Both orthosteric and allosteric modulatory activity has been demonstrated in biochemical validation assays. Allosteric activity was confirmed in cell-based functional assays. The validated fragment hits make excellent starting points for a subsequent hit-to-lead elaboration program.

Functional Insights from Glutamate Receptor Ion Channel Structures

X-ray crystal structures for the soluble amino-terminal and ligand-binding domains of glutamate receptor ion channels, combined with a 3.6-Å-resolution structure of the full-length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of the assembly and function of glutamate receptor ion channels. Increasingly sophisticated biochemical, computational, and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists and suggest new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for the development of subtype-selective modulators. The many unresolved issues include the role of the amino-terminal domain in AMPA receptor signaling and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how glutamate binding opens the ion channel pore.

PAOPA, a potent dopamine D2 receptor allosteric modulator, prevents and reverses behavioral and biochemical abnormalities in an amphetamine-sensitized preclinical animal model of schizophrenia

Allosteric modulators are emerging as new therapeutics for the treatment of psychiatric illnesses, such as schizophrenia. Conventional antipsychotic drugs are typically dopamine D2 receptor antagonists that compete with endogenous dopamine at the orthosteric site, and block excessive dopamine neurotransmission in the brain. However, they are unable to treat all symptoms of schizophrenia and often cause adverse motor and metabolic side effects. The binding profile of allosteric modulators differs, as they interact with their receptor at a novel binding site and their activity is determined by physiological signaling. In collaboration, our laboratories have synthesized and evaluated over 185 compounds for their allosteric modulatory activity at the dopamine D2 receptor. Of these compounds, PAOPA is among the most potent allosteric modulators, and has been shown to be effective in treating the MK-801 induced preclinical animal model of schizophrenia. The objective of this study was to evaluate PAOPA's ability to prevent and reverse behavioral abnormalities in an amphetamine-sensitized preclinical animal model of schizophrenia. Amphetamine sensitized rats were given PAOPA during sensitization and following sensitization to determine whether PAOPA is able to prevent and reverse behavioral abnormalities. Furthermore, changes in post-mortem dopamine levels were measured by high performance liquid chromatography in various brain regions. The results presented demonstrate that PAOPA is able to prevent and reverse behavioral and biochemical abnormalities in an amphetamine-sensitized animal model of schizophrenia.

Reduced brain levels of DHEAS in hepatic coma patients: Significance for increased GABAergic tone in hepatic encephalopathy

Increased neurosteroids with allosteric modulatory activity on GABAA receptors such as 3α–5α tertrahydroprogesterone; allopregnanolone (ALLO), are candidates to explain the phenomenon of “increased GABAergic tone” in hepatic encephalopathy (HE). However, it is not known how changes of other GABAA receptor modulators such as dehydroepiandrosterone sulfate (DHEAS) contribute to altered GABAergic tone in HE. Concentrations of DHEAS were measured by radioimmunoassay in frontal cortex samples obtained at autopsy from 11 cirrhotic patients who died in hepatic coma and from an equal number of controls matched for age, gender, and autopsy delay intervals free from hepatic or neurological diseases. To assess whether reduced brain DHEAS contributes to increased GABAergic tone, in vitro patch clamp recordings in rat prefrontal cortex neurons were performed. A significant reduction of DHEAS (5.81±0.88ng/g tissue) compared to control values (9.70±0.79ng/g, p<0.01) was found. Brain levels of DHEAS in patients with liver disease who died without HE (11.43±1.74ng/g tissue), and in a patient who died in uremic coma (12.56ng/g tissue) were within the control range. Increasing ALLO enhances GABAergic tonic currents concentration-dependently, but increasing DHEAS reduces these currents. High concentrations of DHEAS (50μM) reduce GABAergic tonic currents in the presence of ALLO, whereas reduced concentrations of DHEAS (1μM) further stimulate these currents. These findings demonstrate that decreased concentrations of DHEAS together with increased brain concentrations of ALLO increase GABAergic tonic currents synergistically; suggesting that reduced brain DHEAS could further increase GABAergic tone in human HE.

Two galantamine titration regimens in patients switched from donepezil

In addition to inhibiting acetylcholinesterase, galantamine has allosteric-modulating activity at nicotinic receptors. This may make galantamine an attractive option for patients starting treatment for Alzheimer's disease (AD), but also for those who have not benefited from their current therapy. This study explored outcomes in subjects with AD transitioning from donepezil because of insufficient tolerability or efficacy. Subjects previously receiving donepezil for mild-to-moderate AD were enrolled in a 12-week randomized, open-label study. After screening and a 7-day washout, subjects were randomly allocated to galantamine fast (8 mg/week increments) or slow (8 mg/4 week) titration to 16-24 mg. Efficacy outcomes included the Alzheimer's Disease Assessment Scale - cognitive subscale (ADAS-cog/11), Mini-Mental State Examination (MMSE), Clinician's Interview-Based Impression of Change - Plus Caregiver's Input (CIBIC-plus) and Alzheimer's Disease Cooperative Study - Activities of Daily Living Inventory (ADCS-ADL). Eighty-six of 89 patients (fast titration, n = 44; slow titration, n = 45) completed the study. At week 12, ADAS-cog/11 score improved from screening by 2.6 and 0.6 in the fast- and slow-titration arms, respectively (overall, -1.6; P = 0.002). MMSE scores improved slightly in both arms (overall, +0.9; P = 0.002). Two-thirds of patients had improvement or no change on the CIBIC-plus at week 12. ADCS-ADL scores did not change significantly from screening in either treatment arm. Galantamine was generally well tolerated; nausea (5.6%) and bradycardia (4.5%) were the most commonly reported adverse events. Patients in whom donepezil is ineffective or poorly tolerated may benefit from a switch to galantamine.

4-Benzimidazolyl-3-Phenylbutanoic Acids As Novel Pif-Pocket-Targeting Allosteric Inhibitors of Protein Kinase PKCζ

Protein kinase inhibitors with an allosteric mode of action are expected to reach, in many cases, higher selectivity for the target enzyme than ATP-competitive compounds. Therefore, basic research is aiming at identifying and establishing novel sites on the catalytic domain of protein kinases which might be targeted by allosteric inhibitors. We previously published the first structure-activity relationships (SARs) for allosteric activators of protein kinase PDK1. Here, we present the design, synthesis, and SAR data on a series of novel compounds, 4-benzimidazolyl-3-phenylbutanoic acids, that inhibit the atypical protein kinace C (PKC) ζ via binding to the PIF-pocket. Key positions were identified in the compounds that can be modified to increase potency and selectivity. Some congeners showed a high selectivity toward PKCζ, lacking inhibition of the most closely related isoform, PKCι, and of further AGC kinases. Furthermore, evidence is provided that these compounds are also active toward cellular PKCζ without loss of potency compared to the cell-free assay.

BclxL Changes Conformation upon Binding to Wild-type but Not Mutant p53 DNA Binding Domain

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

Allosteric Modulation of the Dopamine D2 Receptor by Pro-Leu-Gly-NH2 Peptidomimetics Constrained in Either a Polyproline II Helix or a Type II β-Turn Conformation

Type II beta-turn mimics and polyproline II helix mimics based upon diastereoisomeric 5.6.5 spiro bicyclic scaffolds have provided two pairs of Pro-Leu-Gly-NH(2) (PLG) peptidomimetics with contrasting dopamine receptor modulating activities. Compounds 1a and 3a were found to be positive allosteric modulators of the dopamine receptor, while the corresponding diastereoisomeric compounds 1b and 3b provided the first PLG peptidomimetics with the ability to decrease the binding of agonists to the dopamine receptor. The positive allosteric modulating activity of 3a supported the hypothesis that a polyproline II helix conformation is the bioactive conformation for the PLG analogue Pro-Pro-Pro-NH(2). The results also show that a change in the bridgehead chirality of the 5.6.5 scaffold brings about opposite effects in terms of the modulation of the dopamine receptor.

Transposition of Three Amino Acids Transforms the Human Metabotropic Glutamate Receptor (mGluR)-3-Positive Allosteric Modulation Site to mGluR2, and Additional Characterization of the mGluR2-Positive Allosteric Modulation Site

Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.

A Novel Family of Potent Negative Allosteric Modulators of Group II Metabotropic Glutamate Receptors

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.

Synthesis and biological activity of allosteric modulators of GABA B receptors part 3. 3-(2,6-bis-iso-propyl-4-hydroxyphenyl)propanols

A series of six 2,2-disubstituted 3-[3,5-di-iso-propyl-4-hydroxyphenyl]propan-1-ol derivatives have been prepared for evaluation as allosteric modulators of GABAB receptors. The activity (EC50 30 µM) was greatest for the dimethyl analogue, but the isopropylphenyl compounds were generally weaker than the corresponding t-butyl compounds. Methylation of the phenolic group led to loss of activity.

A Region in the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.

The allosteric potentiation of nicotinic acetylcholine receptors by galantamine is transduced into cellular responses in neurons: Ca2+ signals and neurotransmitter release.

Neuronal nicotinic acetylcholine receptors (nAChR) modulate a variety of cellular responses, including Ca2+ signals and neurotransmitter release, which can influence neuronal processes such as synaptic efficacy and neuroprotection. In addition to receptor activation through the agonist binding site, an allosteric modulation of nAChR has also been described for a novel class of allosteric ligands. Of these, the acetylcholinesterase inhibitor and Alzheimer drug galantamine represents the prototypical allosteric ligand, based on its potentiation of nAChR-evoked single-channel and whole-cell currents. The aim of this study was to establish whether the allosteric potentiation of nAChR currents is transduced in downstream cellular responses to nAChR activation, namely increases in intracellular Ca2+ and [3H]noradrenaline release. In SH-SY5Y cells, galantamine potentiated nicotine-evoked increases in intracellular Ca2+ and [3H]noradrenaline release with a bell-shaped concentration-response profile; maximum enhancement of nicotine-evoked responses occurred at 1 muM galantamine. This potentiation was blocked by mecamylamine, whereas galantamine had no effect on these measures in the absence of nicotine. Galantamine did not compete for radioligand binding to the agonist binding sites of several nAChR subtypes, consistent with an allosteric mode of action. Unlike galantamine, the acetylcholinesterase inhibitors rivastigmine and donepezil did not potentiate nAChR-mediated responses, whereas donepezil was a reasonably potent inhibitor of nicotine- and KCl-evoked increases in Ca2+. nAChR-mediated [3H]noradrenaline release from hippocampal slices was also potentiated by galantamine, with an additional component attributable to acetylcholinesterase inhibition and subsequent increase in acetylcholine. These results indicate that the allosteric regulation of nAChR results in the potentiation of receptor-dependent cellular processes relevant to many of the physiological consequences of nAChR activation.

Pharmacokinetic rationale for switching from donepezil to galantamine

Galantamine, the most recently approved acetylcholinesterase inhibitor (AChEI) for usein the United States, has allosteric modulating activity at nicotinic receptors and inhibits acetylcholinesterase. This dual mechanism of action may make galantamine an attractive option for patients with Alzheimer's disease who have not benefited from their current therapy; thus, methods for switching patients from donepezil or rivastigmine to galantamine are needed. Protocols for switching patients from one AChEI to another must consider both the time required for washout of the first drug and the rate of dose escalation of the second drug. Both issues depend on the pharmacodynamics, pharmacokinetics, and pharmacology of the drugs under consideration. Because the common property of the drugs considered here is their acetylcholinesterase inhibitory activity, it seems reasonable to keep this activity at or below the activity achieved by the first drug at all times. In addition, the patient's condition should be monitored to avoid deterioration resulting from subtherapeutic drug concentrations during the switch. The switching protocol proposed here has been based on an analysis of mean plasma concentrations of donepezil following administration of a single dose and on the established pharmacokinetics of galantamine.

Pharmacokinetic profiles of current therapiesfor Alzheimer's disease: implications for switching to galantamine

Although no pharmacologic treatments have been proved to alter the pathology of Alzheimer's disease, acetylcholinesterase inhibitor (AChEI) therapy offers symptomatic improvements in or delays in the progression of cognitive, behavioral, and functional deficits. Tacrine, donepezil, rivastigmine, and galantamine are the best studied agents in this class. These drugs have varying pharmacokinetic, safety, and tolerability profiles that can affect patient outcomes. Specifically, certain metabolic parameters (ie, half-life and route of metabolism/elimination) can affect a drug's tolerability and become important when a switch from one agent to another is contemplated. The mechanism of action of galantamine, the most recently approved AChEI, varies from that of the other AChEIs in that it has allosteric modulating activity at nicotinic receptors in addition to its ability to inhibit acetylcholinesterase. Benefits of this dual mode of action on the effects of the disease have been suggested.

Competitive and allosteric interactions of 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11 h-dibenzo[b,e][1, 4]diazepine-11-one hydrochloride (UH-AH 37) at muscarinic receptors, via distinct epitopes

6-Chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11 H-dibenzo[ b, e][1,4]diazepine-11-one hydrochloride (UH-AH 37) is an analog of pirenzepine that has previously been reported to interact with classical muscarinic antagonists in a competitive manner, yet its binding has also been found to be sensitive to the same epitope as is that of the allosteric ligand gallamine. The present study was carried out with wild-type and chimeric muscarinic receptors to determine whether UH-AH 37 might also have an allosteric mode of action. In assays that detect only allosteric interactions, UH-AH 37 slowed the rate of dissociation of [ 3H] N-methylscopolamine (NMS) from all five muscarinic receptor subtypes, with the highest apparent affinity at m2. By contrast, studies carried out under equilibrium conditions have found UH-AH 37 to have the lowest affinity for the m2 subtype. Studies with m2/m5 chimeric receptors found the allosteric potency of UH-AH 37 to be sensitive to an epitope in the seventh transmembrane domain (TM). Again, this contrasts with equilibrium studies, wherein an epitope in the sixth TM has been implicated. Simultaneous analysis of the interactions between UH-AH 37 and [ 3H]NMS at the m2 receptor under equilibrium and non-equilibrium conditions found that a simple allosteric model could not accommodate both sets of data. On the other hand, the model did accommodate such data for gallamine; gallamine also displays concordance in order-of-potency and epitope sensitivity between equilibrium and non-equilibrium assays. Based on these results, we conclude that UH-AH 37 interacts at the classical muscarinic binding site with high affinity and at a second (allosteric) site with lower affinity.

On the analysis of futile cycles in metabolism

So-called futile cycles in cellular metabolism consist of paired opposing reactions that, if simultaneously operant, act only to degrade free energy of ATP to heat. Previous considerations of the behavior of such substrate cycles have indicated their possible usefulness in regulating flux along metabolic pathways, but such analyses have treated the cycles in isolation, i.e. without taking into account the effects of enzymatic inputs to and outputs from the cycle. We here develop models of three typical substrate cycles that include enzymatic inputs to and outputs from the cycle and allow the enzymes of the cycles per se to be subject to a variety of allosteric modulations. The non-linear equations which describe these models were solved by an iterative procedure for sets of parameter values of metabolic interest. The results, when analyzed using appropriate definitions of regulatory sensitivity and energetic futility, demonstrate that the effects of the enzymes leading into and out of the cycle may cause profound changes in the operation of the substrate cycle and therefore may not be ignored. We find that the structural differences among the three cycles considered here result in corresponding functional differences. Our results suggest that (1) the fructose-6-P/fructose-1,6-di-P cycle acts effectively to gate bidirectional flux, but doesn't appreciably enhance regulation of unidirectional flux, (2) the glucose/glucose-6-P cycle is well suited to perform a homeostatic function and to adjust the set points for these two metabolites, and (3) the cycle at the pyruvate crossroads functions largely as a complex switch box that directs metabolic flow towards gluconeogenesis or glycolysis not only in response to inputs of or requirements for oxaloacetate, pyruvate, and phosphoenolpyruvate, but also in response to the combined action of allosteric modulators on the individual enzymes of this substrate cycle.