A clickable analogue of ketamine retains NMDA receptor activity, psychoactivity, and accumulates in neurons

Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamine’s psychoactive effects, ketamine or its major metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel visualizable analogues of ketamine/norketamine. We introduced an alkyne “click” handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom. Ketamine, norketamine, and A-NK, but not A-NK-amide, showed acute and persisting psychoactive effects in mice. This psychoactivity profile paralleled activity of the compounds as NMDAR channel blockers; A-NK-amide was inactive at NMDARs, and norketamine and A-NK were active but ~4-fold less potent than ketamine. We incubated rat hippocampal cells with 10 μM A-NK or A-NK-amide then performed Cu(2+) catalyzed cycloaddition of azide-Alexa Fluor 488, which covalently attaches the fluorophore to the alkyne moiety in the compounds. Fluorescent imaging revealed intracellular localization of A-NK but weak A-NK-amide labeling. Accumulation was not dependent on membrane potential, NMDAR expression, or NMDAR activity. Overall, the approach revealed a correlation among NMDAR activity, intracellular accumulation/retention, and behavioral effects. Thus, we advance first generation chemical biology tools to aid in the identification of ketamine targets

State-Dependent Mapping of GlyR-Cholesterol Interactions by Coupling Crosslinking with Mass Spectrometry

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol: lipid ratios below and above the cholesterol activity threshold with aliquots of azi-cholesterol. State-dependent crosslinking studies of receptors primarily in its resting (no glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM ivermectin) states were then performed at elevated cholesterol levels necessary for activity. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spectrometry (MS-MS), and sites of cholesterol crosslinks in peptides were refined by targeted MS-MS. Within the GlyR apo state, cholesterol interactions differed as a function of membrane cholesterol concentration correlating to the chemical activity of cholesterol, suggesting two distinct conformations. Differential cholesterol crosslinking patterns between resting, desensitized, and open states were observed, highlighting state-dependent differences in GlyR lipid accessibility. Distinct state-dependent crosslinking patterns indicative of alterations in either the lipid environment and/or channel structure were observed throughout GlyR, most prominently observed in the M4 transmembrane helix, extracellular domain loops and regions nearing the bilayer interface, and the large intracellular M3-M4 loop. The changes in M4 accessibility (transition from surface-mapped crosslinking to regions of the helix less exposed when mapped) suggest an outward twisting motion and translocation towards the bilayer/lipids as GlyR allosterically transitions. Strikingly, crosslinking patterns within the M3-M4 loop offer insight into the generalized structure of this unresolved region in all current pLGIC structural models, by suggesting the crosslinked regions of this intracellular loop are intimately associated or buried within the lipid bilayer. Taken together, crosslinking coupled with MS-MS has the capability to accurately probe and define physiological protein frameworks which can aid in the refinement of allosteric modulation and current structural models

Understanding The Structure-Function Relationships Between Monoamine Neurotransmitter Transporters And Their Cognate Ions And Ligands

The SLC6 family of secondary active transporters is made up of integral membrane solute carrier proteins characterized by the Na+-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, particularly the monoamine transporters (MATs) of serotonin, dopamine and norepinephrine, are some of the most heavily studied proteins today due to their association with a number of human diseases and disorders, making MATs a critical target for therapeutic development. In addition, MATs are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Following the first cloning of a MAT gene in the early 1990s, much has been uncovered about the structure and function of these proteins. Early studies developed an understanding of the kinetic parameters by which MATs operate and also yielded enough information to model the basic structural characteristics of MATs. This was greatly improved upon within the last decade, as crystallographic and computational advances have provided structural insights that have vastly accelerated our ability to study these proteins and their involvement in complex biological processes. However, despite a wealth of knowledge concerning the structural and kinetic characteristics of MATs, little is understood as to how these features are interrelated and much is still unclear as to the how regulation (and maybe more importantly, dysregulation) of MATs alters the functionality of these proteins at the molecular and synaptic levels. The overall goal of this dissertation was to comprehensively examine the relationship between MAT structure and the ions and ligands that bind to MATs to promote/prevent transporter function. This was done using a comprehensive approach that included biological, electrophysiological and computational techniques to target and elucidate the roles of specific amino acid residues in ion/ligand binding and/or mediation of the substrate translocation process. In successfully examining a number of specific MAT residues, this work has lead to the deduction of basic roles for each of the ion binding sites in the translocation mechanism (chapters II and III), as well as detailed the importance of specific structural components of MATs that are vital for functionality (chapters IV and V). Furthermore, this dissertation includes work highlighting the development of several photo-labeled, radio-iodinated antagonist analogues that will be used to further improve the understanding of how inhibitors bind to and block MAT function at the molecular level (chapter VI). In total, the work outlined in this dissertation provides a clearer understanding as to the molecular interactions that are necessary for MAT function and contributes an improved appreciation for the underlying mechanisms of substrate translocation and pharmacological intervention

Characterization Of Dopamine Transporter Amino Terminal Palmitoylation And Neurotoxic Substrate Activity

The projects described in this dissertation revolve around the functional consequences experienced by the transport protein for the neurotransmitter dopamine resulting from alterations to its palmitoylation condition or exposure to Parkinson disease-inducing transport substrates. This membrane-resident transport protein, pragmatically termed the dopamine transporter, relocates dopamine from extracellular areas of receptor sites of action to intracellular sequestration; the dopamine transporter is a powerful mediator of dopamine signaling. As such, genetic, toxicant, or chronic breakdown of dopamine transporter function is associated with multiple psychological abnormalities. Biomedical research has produced several pharmacotherapies for maladies like depression, attention deficit/hyperactivity disorder, and addiction, some of which produce their therapeutic profiles by modulating dopamine transporter function. The potential for pharmacological manipulation of the dopamine transporter is, however, not without its dark side. Dopamine’s critical role in the neurotransmission of reward and pleasure render the dopamine transporter a favorite target of illicit, addictive drugs of abuse like cocaine and several flavors of amphetamine. All of these factors make intimate understanding of the many mechanisms involved in the dopamine transporter’s function relevant not only to mental, but also societal health. This dissertation explores aspects of dopamine transporter mechanistic regulation which, once more thoroughly understood, may be modified to allow finer control over transporter operation, generating novel approaches to mental health treatment. The first study investigates site identification and functional characterization of post-translational modification of the dopamine transporter by a lipid moiety, palmitic acid. Palmitic acid, hexadecanoic acid in IUPAC nomenclature, is a saturated 16 carbon fatty acid whose attachment to proteins is termed S-palmitoylation. This lipidation process is executed by an array of enzymes belonging to the acyl transferase class of the gene name zDHHC. Importantly, S-palmitoylation is reversible: a protein’s palmitoylation status can change in response to cell stimuli or the palmitoylated protein’s activation. As palmitate is of an aliphatic nature, its attachment creates a hydrophobic protein microenvironment around the site of its augmentation which propitiates its insertion into likewise hydrophobic loci – usually membranes – which induces a protein-specific functional outcome. A combination of dopamine transporter proteolysis, site-directed mutagenesis, acyl-biotinyl exchange, surface biotinylation, and forward and reverse dopamine transport assays implicate two N-terminal cysteine residues as sites of palmitate incorporation, in addition to the previously analyzed C-terminal site, and reveal a role for this lipid modification in dopamine transporter-mediated dopamine efflux. The second project seeks to further understand the dopamine transporter’s contribution to Parkinson disease. The hallmark of this disease is a loss of motor coordination precipitated by selective death of nigro-striatal dopamine neurons and concomitant depletion of dopamine neurotransmission in the movement planning and execution region of the brain – the striatum. The selective loss of these neurons directly correlates with dopamine transporter expression; indeed, even amongst dopamine neuronal pathways, the nigro-striatal fiber, which is lost to the greatest extent of these, has the highest transporter expression. It is for this reason the dopamine transporter has been a focus of Parkinson disease research. This study utilizes the dopamine transporter substrates 6-hydroxydopamine and 1-methyl-4-phenylpyridinium, which induce cell death through a panoply of biochemical mechanisms and are used to generate Parkinsonian symptoms in animal models, to probe for aberrant dopamine transporter function and post-translational modification. This inquiry revealed that, though these compounds induce cell death through similar mechanisms, their dopamine transporter-specific effects are quite different. Interestingly, 1-methyl-4-phenylpyridinim is a strong inducer of dopamine uptake downregulation and dopamine efflux, a phenomenon now implicated in Parkinson disease onset, while 6-hydroxydopamine mitigates this efflux event as well as attenuates transporter phosphorylation. Overall, this dissertation argues for the existence of N-terminal palmitoylation of the dopamine transporter, that palmitoylation is an additional contributor to the dopamine efflux paradigm, that transporter-mediated efflux may contribute to Parkinson disease onset, and that some of the transporter-specific effects of 6-hydroxydopamine may be exploited to alleviate neuropsychiatric maladies associated with aberrant dopamine efflux

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Development of novel carriers for transdermal delivery of peptides

Recent developments in genetic engineering and biotechnology have resulted in anincrease in availability of therapeutic peptides and small anti-cytokines. Oraladministration is inappropriate as these molecules are unstable in the gastrointestinaltract and are subject to hepatic first-pass effect. Transdermal delivery is an attractivealternative as the skin exhibits less enzymatic activity and allows for a controlled,sustained local therapeutic drug concentration over a prolonged period of time.However, the skin’s lipophilic stratum corneum acts as a major barrier to thedelivery of hydrophilic molecules, including peptides, resulting in lack of efficacy ofthese compounds if applied topically. Considerable research effort has beenfocussed on the development of skin penetration enhancement techniques. However,many of these techniques have been limited by insufficient penetration enhancementand/or induced irritancy.We have investigated three approaches to enhance the delivery of peptides that havetherapeutic or cosmetic effect in the skin. These approaches include the use ofphysical energy to enhance the delivery of Alanine-Tryptophan (Ala-Trp), lipoaminoacid (LAA) conjugation to increase the permeability of a HNE inhibitor Ala-Ala-Pro-Val (AAPV) and a cosmetic peptide, acetyl hexapeptide-3 and cyclisation to enhancethe delivery of a core peptide (CP) which has anti-inflammatory activity.In vitro permeation studies across human epidermis were performed in Pyrex glassFranz-type diffusion cells. Ala-Trp was selected as a small molecular weight modeldipeptide to study the penetration enhancement effects of Dermaportation, which is anewly developed pulsed electro magnetic field (PEMF) technology. The dipeptidewas found to be unstable on exposure to skin at 37°C and Dermaportation (Pulsedelectromagnetic field technology) significantly increased the in vitro permeabilitycoefficient of Ala-Trp across human epidermis from 7.7 x 10-4 cm/h with passivediffusion to 1.94 x 10-2 cm/h with Dermaportation over an 8 h period.Dermaportation thus may provide an effective means of delivering molecules that arehighly susceptible to degradation like dipeptides, in higher amounts and in arelatively short duration.The effectiveness of coupling a short chain lipoamino acid to enhance transepidermaldelivery of a model human neutrophil elastase (HNE) inhibitor (Ala-Ala-Pro-Val) was assessed. The optimal conjugate structure for skin penetration andbiological activity of this therapeutic peptide with anti-inflammatory activity wasdetermined. In order to enhance the trans-epidermal delivery of the peptide,lipophilic derivatives with LAAs of chain length C6, C8, and C10 were prepared bysolid phase synthesis. Conjugation to a C6-LAA enhanced epidermal permeability ofthe tetrapeptide. Stereoselective permeation of the lipopeptide diastereomers acrossthe human epidermis was observed. The amount of C6(D)-LAA-AAPV (467.94μg/cm2) was significantly higher than C6(L)–LAA-AAPV (123.04 μg/cm2). Thesame was observed with C8(D)-LAA-AAPV. The effect of donor concentration andskin hydration on skin permeability of C8(D,L)-LAA-AAPV and C10(D,L)-LAAAAPVwas also assessed and it was observed that there was higher permeation ofC10(D,L)-LAA-AAPV at a higher donor concentration. The lipoamino acid conjugates were more stable than the native tetrapeptide and biological activity was retained after coupling of the tetrapeptide to C6, C8 and C10 LAA.A cosmetic peptide, acetyl hexapeptide-3 was coupled to individual diastereomers ofC12 (A)-LAA and C12 (B)-LAA. The preliminary study was designed to assess theeffect of coupling of a LAA of higher molecular weight on the transepidermalpermeation and accumulation of this hexapeptide. Accumulation of these peptides inthe skin was also quantified. Detectable amounts of C12(A)-LAA-hexapeptide-3 andC12(B)-LAA-hexapeptide-3 were not found in the receptor solution but higherquantities of these conjugates were found to be retained in the skin. The amount ofC12(B)-LAA-hexapeptide-3 (59.92 μg/cm2 ± 10.64) in the epidermis was highestfollowed by C12(A)-LAA-hexapeptide-3 (33.06 μg/cm2 ± 3.70) and acetylhexapeptide-3 (12.64 μg/cm2 ± 1.48).Lastly, skin permeability and in skin stability of an anti-inflammatory peptide (corepeptide: CP) and two analogues that have demonstrated improved biological efficacyand specificity: a cyclic peptide sequence (C1) and its linear sequence counterpart(C1-L) were assessed. The stability of C1 and C1-L was significantly higher ascompared to CP when placed in contact with skin at 37ºC. The epidermal penetrationof the core anti-inflammatory peptide improved after cyclisation. The order of Lastly, skin permeability and in skin stability of an anti-inflammatory peptide (corepeptide: CP) and two analogues that have demonstrated improved biological efficacyand specificity: a cyclic peptide sequence (C1) and its linear sequence counterpart(C1-L) were assessed. The stability of C1 and C1-L was significantly higher ascompared to CP when placed in contact with skin at 37ºC. The epidermal penetrationof the core anti-inflammatory peptide improved after cyclisation. The order of permeation of the analogues was C1>C1-L>CP after 48h and 6 days. The amount of peptide retained in the skin was higher after 48h as compared to 8h due to greater partitioning of these peptides in the skin.This work demonstrates the enhancement effects of these three techniques tooptimize the transdermal/topical permeation of therapeutic and cosmetic peptides

Structural modification of CNG channels during activation

Cyclic nucleotide-gated ion channels are distributed most widely in the neuronal and nonneuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in recent years since their discovery in 1985(Fesenko et al., 1985). Results of many experiments have indicated that the stoichiometry and assembly of CNG channel subunit affect their property and gating. The substituted cysteine accessibility method (SCAM) has been a very powerful tool in understanding many of the molecular mechanisms underlying their functions. Cite directed mutagenesis has been a great help in elucidating the possible mechanism behind the ligand discrimination among channels expressed in different cell types. In the recent years the advance in computer technology has provided tremendous help in understanding the three dimensional arrangement of proteins by virtue of molecular biology. Most probably this is the perfect time in which molecular biology, biochemistry and computational science has come together to provide some amazing view of membrane proteins. Still crystallography has its own limitations, and electrophysiology serves as an adequate substitute. Most of our understanding about the CNG channels arises from the study of these channels expressed in sensory neurons, viz photoreceptors and olfactory sensory neurons. In my work I have used heterologously expressed homologous CNGA1 subunit from bovine rod receptors as a target. The expression system used was Xenopus leavis oocytes. Though the homologous channels thus expressed vary in several aspects provides a very good tool in studying the structure function relationship of these channels. In the preliminary part of the study an extensive site directed mutagenesis from residue F375 to V424, one at a time, has been performed (SCAM). I have then probed these mutant channels with divalent cations such as Cd2+ and Ni2+ and several methane thiosulfonate compounds to study their accessibility and interaction. The residues from F375 until S399 does not show much effect to these externally applied compounds with few exceptions. One remarkable exception is F380C, which is found to be potentiated by Cd2+ when applied in the open state of the channel inhibited when applied in the closed. Further studies have revealed a locking effect of the channel and thus some insight into the proximity of residues and possible molecular rearrangement while channel passing from closed to open. Another study has revealed the interaction of native Cys505 residues with several other residues in the C-linker domain when are mutated into cysteine. This study has helped to propose a molecular model of C-linker domain. Also it provided some knowledge in the possible rearrangement of C-linker region while channel opens. One another course of study has revealed that the residues from 390 to 400 come closer in the closed state than in the open. The following stretch of residues, from 410 to 420, on the contrary comes closer in the open state than in the closed. My studies suggest that the channel while passing from close to open does not undergo a major translational movement of residues near the S6. Probably the coupled movement of of S6 with pore helix provides enough energy to open the gate

MODULATION OF THE RECEPTOR GATING MECHANISM AND ALLOSTERIC COMMUNICATION IN IONOTROPIC GLUTAMATE RECEPTORS

Ionotropic glutamate receptors (iGluRs) found in mammalian brain are primarily known to mediate excitatory synaptic transmission crucial for learning and memory formation. The family of iGluRs consists of AMPA receptors, NMDA receptors and kainate receptors with each member having distinct physiological role. In the recent years, significant progress has been made in understanding the biophysical, and functional properties of iGluRs. The development of Cryo-EM and X-Ray crystallography techniques have further facilitated in the structural understanding of these receptors. However, the multidomain nature, large size of the protein, complex gating mechanism and inadequate knowledge regarding the conformational dynamics of the receptors during channel gating mechanism have been some of the limiting factors in elucidating the structure-function relation of iGluRs. Thus, to understand the conformational dynamics of iGluR family and correlate to its functional behavior, I have utilized single molecule Forster Resonance Energy Transfer (smFRET) and molecular dynamics simulation and specifically investigated the factors influencing gating mechanism and allosteric communication in heteromeric kainate receptor GluK2/K5 and NMDA receptor GluN1/N2A. Some of the major finding in this dissertation includes subunit arrangement of GluK2/K5 and its dynamics involved in resting and desensitized conditions. For the first time we have identified the conformational changes induced at GluK2 and GluK5 subunits in a heteromer GluK2/K5 when bound to different agonists. Utilizing MD simulations in GluN1/N2A NMDA receptors we have identified the structural pathway regarding the mechanism underlying negative cooperativity and how mutation in the receptor leads to abnormal functional behavior. These findings will allow us to understand the conformational control regarding modulation of receptor function and will serve as a basis for developing subunit and conformation-specific therapeutic drugs that can potentially control the abnormal activity of the receptors linked to several neurological diseases

Proceedings of the Merck & Elsevier Young Chemists Symposium (MEYCS 2018)

Dear participants, welcome to the 18th edition of the Merck & Elsevier Young Chemists Symposium, formerly SAYCS and MYCS. This conference is an international scientific event organized by the Young Group of the Italian Chemical Society (SCI Giovani) with the financial support of Merck and Elsevier. This symposium is fully devoted to young researchers, such as MSc and PhD students, post-doc fellows and young researchers in companies. All the disciplines of Chemistry are covered: analytical, physical, industrial, organic, inorganic, theoretical, pharmaceutical, biological, environmental, macromolecular and electrochemistry. This year, a special emphasis will be given to chemistry from knowledge to innovation: how chemistry is increasingly present in all of the fields that are essential for human life, and how chemical fundamentals are pushing novel technologies? This year we have the exceptional number of 212 participants; we thank you for the great trust shown towards SCI Giovani, Merck and Elsevier. Enjoy the conference