Membrane-permeant Molecule Research Articles

Regulation of Cannabinoid and Opioid Receptor Levels by Endogenous and Pharmacological Chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), δ opioid receptor (DOR), and CB1R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.

Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice

Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases.

Converting polar cyclic peptides into membrane permeable molecules using N‐methylation

AbstractDescribed are the design, synthesis, and biological evaluation of 5 N‐methylated analogs that are based on a lead drug structure LB51. LB51 is a cyclic pentapeptide that inhibits heat shock protein 90 and although a potent inhibitor of the protein function, it has poor cell permeability. Introduction of an N‐methyl moiety at each amino acid produces 5 analogs of LB51, where all 5 show significantly improved membrane permeability over the lead molecule despite the presence of 4 highly polar side chains.

Small membrane permeable molecules protect against osmotically induced sealing of t-tubules in mouse ventricular myocytes.

Cardiac t-tubules are critical for efficient excitation-contraction coupling but become significantly remodeled during various stress conditions. However, the mechanisms by which t-tubule remodeling occur are poorly understood. Recently, we demonstrated that recovery of mouse ventricular myocytes after hyposmotic shock is associated with t-tubule sealing. In this study, we found that the application of Small Membrane Permeable Molecules (SMPM) such as DMSO, formamide and acetamide upon washout of hyposmotic solution significantly reduced the amount of extracellular dextran trapped within sealed t-tubules. The SMPM protection displayed sharp biphasic concentration dependence that peaks at ∼140 mM leading to >3- to 4-fold reduction in dextran trapping. Consistent with these data, detailed analysis of the effects of DMSO showed that the magnitude of normalized inward rectifier tail current (IK1,tail), an electrophysiological marker of t-tubular integrity, was increased ∼2-fold when hyposmotic stress was removed in the presence of 1% DMSO (∼140 mM). Analysis of dynamics of cardiomyocytes shrinking during resolution of hyposmotic stress revealed only minor increase in shrinking rate in the presence of 1% DMSO, and cell dimensions returned fully to prestress values in both control and DMSO groups. Application and withdrawal of 10% DMSO in the absence of preceding hyposmotic shock induced classical t-tubule sealing. This suggests that the biphasic concentration dependence originated from an increase in secondary t-tubule sealing when high SMPM concentrations are removed. Overall, the data suggest that SMPM protect against sealing of t-tubules following hyposmotic stress, likely through membrane modification and essentially independent of their osmotic effects.

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors

BackgroundSmall membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized.MethodsCytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated.ResultsCHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T.ConclusionsOut of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.

オリゴアルギニン固定化高分子を用いた低膜透過性分子の細胞膜透過促進

オリゴアルギニン固定化高分子を用いた低膜透過性分子の細胞膜透過促進

Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes

We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.

Specific ligands as pharmacological chaperones: The transport of misfolded G‐protein coupled receptors to the cell surface

In the endoplasmic reticulum (ER), quality control mechanisms distinguish between correctly and incorrectly folded structures to ensure that aberrant proteins are not processed along the secretory pathway. Numerous studies have demonstrated the functional rescue of ER-retained, aberrant proteins by small membrane permeable molecules called pharmacological chaperones. Pharmacological chaperones can bind to misfolded proteins, including G-protein coupled receptors (GPCRs), and promote their correct folding and export from the ER. Recently, common structural features of GPCRs have been uncovered, including the eighth helical domain in the C-terminal tail and conserved residues in the transmembrane domains. However, little is known about the importance of these features in signaling and intracellular trafficking, because receptors deficient in these domains are likely retained in the ER due to misfolding. In this review, we summarize the current knowledge about the requirement of these consensus domains and amino acid residues for the passing through the quality control of the ER. Furthermore, we propose the utilization of membrane permeable ligands for the transport of their cognate, ER-retained GPCRs to the cell surface. The chaperone activity of these ligands allows us to perform functional analyses of the structure-deficient receptors after their trafficking to the cell surface.

Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo -inositol trispyrophosphate

A major determinant of maximal exercise capacity is the delivery of oxygen to exercising muscles. myo-Inositol trispyrophosphate (ITPP) is a recently identified membrane-permeant molecule that causes allosteric regulation of Hb oxygen binding affinity. In normal mice, i.p. administration of ITPP (0.5-3 g/kg) caused a dose-related increase in the oxygen tension at which Hb is 50% saturated (p50), with a maximal increase of 31%. In parallel experiments, ITPP caused a dose-related increase in maximal exercise capacity, with a maximal increase of 57 +/- 13% (P = 0.002). In transgenic mice with severe heart failure caused by cardiac-specific overexpression of G alpha q, i.p. ITPP increased exercise capacity, with a maximal increase of 63 +/- 7% (P = 0.005). Oral administration of ITPP in drinking water increased Hb p50 and maximal exercise capacity (+34 +/- 10%; P < 0.002) in normal and failing mice. Consistent with increased tissue oxygen availability, ITPP decreased hypoxia inducible factor-1alpha mRNA expression in myocardium. It had no effect on myocardial contractility in isolated mouse cardiac myocytes and did not affect arterial blood pressure in vivo in mice. Thus, ITPP decreases the oxygen binding affinity of Hb, increases tissue oxygen delivery, and increases maximal exercise capacity in normal mice and mice with severe heart failure. ITPP is thus an attractive candidate for the therapy of patients with reduced exercise capacity caused by heart failure.

Using BioTek's Synergy™ HT Reader to Measure Reactive Oxygen Species (ROS) Generation in Stimulated Cells

The investigation of biological agents that can stimulate or inhibit cell growth is of great interest to cellular biologists. One such measure is the formation of Reactive Oxygen Species (ROS) as a result of cell stimulation. While there are numerous methodologies to assess ROS species, the use of fluorescent probes has become the preferred method. Here we describe the measurement of DCF fluorescence using a SynergyTM HT to detect and analyze the production of ROS as a result of cell growth stimulation.

Sox11 transcription factor modulates peripheral nerve regeneration in adult mice

The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.

Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3′-termini

Short-interfering RNAs (siRNAs) conjugated with lipophilic groups at their 3′-termini were synthesized. The properties of the synthesized siRNAs were examined in detail, and it was found that at low concentrations, their silencing abilities were dependent on the positions of the modifications and the types of organic molecules attached. Although the modification of siRNAs with palmitic acid or oleic acid at the 3′-end slightly reduced their silencing activities, siRNAs had enough abilities to induce RNAi at 10 nM concentrations. On the other hand, the modification of siRNAs with cholesterol at the 3′-end of the passenger strand was tolerated; however, the modification at the guide strand significantly reduces its silencing activity. The siRNAs modified with the lipophilic groups did not possess ability to penetrate the plasma membranes of HT-1080 cells without the transfection reagent. However, the results described in this report will aid in designing novel siRNAs with cell membrane-permeable molecules.

Pharmacological approaches to nitric oxide signalling during neural development of locusts and other model insects

A novel aspect of cellular signalling during the formation of the nervous system is the involvement of the messenger molecule nitric oxide (NO), which has been discovered in the mammalian vascular system as mediator of smooth muscle relaxation. NO is a membrane-permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cell types in model insects such as Locusta, Schistocerca, Acheta, Manduca, and Drosophila shows that the NO/cGMP pathway is required for the stabilization of photoreceptor growth cones at the start of synaptic assembly in the optic lobe, for regulation of cell proliferation, and for correct outgrowth of pioneer neurons. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO, and potentially also another atypical messenger, carbon monoxide (CO), orchestrate cell migration of enteric neurons. Cultured insect embryos are accessible model systems in which the molecular pathways linking cytoskeletal rearrangement to directed cell movements can be analyzed in natural settings. Based on the results obtained from the insect models, I discuss current evidence for NO and cGMP as essential signalling molecules for the development of vertebrate brains.

STOP and GO with NO: Nitric oxide as a regulator of cell motility in simple brains

During the formation of the brain, neuronal cell migration and neurite extension are controlled by extracellular guidance cues. Here, I discuss experiments showing that the messenger nitric oxide (NO) is an additional regulator of cell motility. NO is a membrane permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cells types in invertebrate models such as molluscs, insects and the medicinal leech provides insight how NO and cyclic nucleotides affect the wiring of nervous systems by regulating cell and growth-cone motility. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO signalling orchestrates neurite outgrowth and filopodial dynamics, cell migration of enteric neurons, glial migration and axonogenesis of pioneer fibers. Cultured insect embryos are accessible model systems in which cellular mechanisms of NO-induced cytoskeletal reorganizations can be analyzed in natural settings. Finally, I will outline some indications that NO may also regulate cell motility in the developing and regenerating vertebrate nervous system.

Estrogen Barges In

The steroid hormone estrogen acts both through nuclear receptors that control transcription of target genes and through signaling pathways outside the nucleus. Revankar et al. (see the Perspective by Hewitt et al. ) report that a G protein-coupled receptor located in the membrane of the endoplasmic reticulum mediates estrogen signaling in various cell types. Upon binding to estrogen, the receptor stimulates mobilization of intracellular calcium and synthesis of nuclear phosphatidylinositol 3,4,5-trisphosphate, both of which trigger further signaling events. Estrogen is a membrane-permeable molecule, and it is likely that its access to intracellular membrane receptors can facilitate some of the rapid nongenomic signaling initiated by the hormone. C. M. Revankar, D. F. Cimino, L. A. Sklar, J. B. Arterburn, E. R. Prossnitz, A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307 , 1625-1630 (2005). [Abstract] [Full Text] S. C. Hewitt, B. J. Deroo, K. S. Korach, A new mediator for an old hormone? Science 307 , 1572-1573 (2005). [Summary] [Full Text]

Sending a Cell-Death Sentence

Cancer cells proliferate because they evade programmed cell death pathways, and much effort is being devoted to find ways to activate apoptotic pathways in such cells (see the Perspective by Denicourt and Dowdy). Key interactions that determine whether cells live or die are mediated by so-called BH3 (BCL-2 homology 3) domains, which are found in proteins that regulate apoptosis. Such signals can be mimicked or disrupted by peptides that resemble the interaction domains, but such molecules have major shortcomings as experimental or therapeutic agents because of low potency, instability, and inefficient delivery to cells. Walensky et al. now show that these problems could be overcome when a BH3 domain that promotes apoptosis was held in its native α-helical form by a chemical modification they call a hydrocarbon staple. The modified peptide showed increased binding affinity for its target, was relatively protease resistant, and could cross cell membranes. Preliminary studies in animals even showed that the modified peptides could decrease growth of transplanted tumors in mice. The activity of caspases, the cysteine proteases that mediate cell death by apoptosis, is held in check by inhibitor of apoptosis proteins (IAPs). The protein known as Smac promotes apoptosis by binding to IAPs and relieving inhibition of caspases. Li et al. show that the effect of the Smac peptide can be potently mimicked by a small membrane-permeable molecule. Studies with the compound revealed that the well-known requirement for inhibition of protein synthesis to allow apoptotic effects of tumor necrosis factor-α (TNF-α) likely reflects decreased IAP-mediated inhibition of caspases. The new compound sensitized cancer cells in culture to TNF-α-induced cell death. L. D. Walensky, A. L. Kung, I. Escher, T. J. Malia, S. Barbuto, R. Wright, G. Wagner, G. L. Verdine, S. J. Korsmeyer, Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305 , 1466-1470 (2004). [Abstract] [Full Text] L. Li, R. M. Thomas, H. Suzuki, J. K. De Brabander, X. Wang, P. G. Harran, A small molecule Smac mimic potentiates TRAIL- and TNFα-mediated cell death. Science 305 , 1471-1474 (2004). [Abstract] [Full Text] C. Denicourt, S. F. Dowdy, Targeting apoptotic pathways in cancer cells. Science 305 , 1411-1413 (2004). [Summary] [Full Text]

Hormone release is tied to changes in cell size in the osmoreceptive prolactin cell of a euryhaline teleost fish, the tilapia, Oreochromis mossambicus

Prolactin (PRL) cells from a teleost fish, the tilapia, Oreochromis mossambicus, facilitate the direct study of osmoreception. The release of two prolactins, PRL 188 and PRL 177, which act in freshwater osmoregulation in teleost fish, rises in vitro within 5 min after extracellular osmolality falls. An increase in cell size accompanied this rise. Cell size and PRL release also increased, albeit more slowly, following the partial replacement of medium NaCl (55 mOsmolal) with an equivalent concentration of urea, a membrane-permeant molecule. Similar replacement using mannitol, which is membrane-impermeant, elicits no response. These findings suggest that osmoreception is linked to changes in cell volume rather than to extracellular osmolality per se.

Rescue of hypogonadotropic hypogonadism-causing and manufactured GnRH receptor mutants by a specific protein-folding template: misrouted proteins as a novel disease etiology and therapeutic target.

In the present study, we demonstrate pharmacological rescue (assessed by ligand binding and restoration of receptor coupling to effector) of five naturally occurring GnRH receptor (GnRHR) mutants (T(32)I, E(90)K, C(200)Y, C(279)Y, and L(266)R), identified from patients with hypogonadotropic hypogonadism, as well as rescue of other defective receptors intentionally manufactured with internal or terminal deletions or substitutions at sites expected to be involved in establishment of tertiary receptor structure. The pharmacological agent used is a small, membrane-permeant molecule, originally designed as an orally active, nonpeptide receptor antagonist, but is believed to function as a folding template, capable of correcting the structural defects caused by the mutations and thereby restoring function. After rescue, this agent can be demonstrably removed. The rescued receptor, now stabilized in the plasma membrane, couples ligand binding to activation of the appropriate effector system. For comparison, low-, intermediate-, or high-affinity peptide antagonists of GnRHR (that do not penetrate the cell) were unable to effect rescue, as was a nonbinding peptidomimetic congener of the rescue agent; this latter effect demonstrates specificity of the rescue agent. Our findings, taken in concert with an earlier study showing rescue of a mutant by modifications to the receptor structure that enhance plasma membrane expression of the GnRHR, suggest that mutant GnRHRs have frequently not lost intrinsic functionality and are subject to rescue by techniques that enhance membrane expression. The present findings demonstrate the efficacy of an approach based on pharmacological rescue and suggest the basis of new approaches for intervention in this and similar diseases.

Special Delivery For Small Molecules

Amicrofluidics technique allows researchers to deliver certain kinds of molecules selectively into regions of a living cell. The work is a collaboration between the groups of Donald E. Ingber, professor of pathology at Harvard Medical School, and Harvard chemistry professor George M. Whitesides [ Nature , 411 , 1016 (2001)]. Using a technique developed previously, the researchers produce parallel and adjacent streams of different fluids that flow laminarly and mix only slowly by diffusion. If the streams contain membrane-permeable molecules, they will flow right through a cell placed in their path, thereby delivering one type of molecule to one part of the cell and another type to another part. In the experiment shown here (at left), a red dye stains mitochondria in the upper half of a bovine capillary endothelial cell, while a green dye stains mitochondria in the lower half of the same cell. The entire cell is also treated with a DNA-staining ...