Preprotein signature for full susceptibility to the co-translational translocation inhibitor cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.

Full Visible Spectrum and White Light Emission with a Single, Input-Tunable Organic Fluorophore.

The blue emission of M2biQ can be tuned to specific wavelengths throughout the visible region by changing the identity of the cation it interacts with. These optical properties are observed in MeCN solution and the solid state. White light is obtained in MeCN by using either the proper ratio of zinc ions or acid. Thus, M2biQ acts as a nearly universal emitter (λem = 468-690 nm) with large Stokes shifts (116-306 nm, Δν̃ = 7,042-11,823 cm-1). Full spectral profiles as well as quantum yields, lifetimes, and the crystal structures of key RGB and yellow emitters are reported. Emission wavelengths correlate with cationic radius, and TD-DFT calculations show that, for 1:1 complexes, the smaller the ion, the shorter the N-cation bond, and the greater the bathochromic emission shift.

The signal peptide as a new target for drug design.

Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to translocon channels across the membrane of the endoplasmic reticulum (ER) by signal peptides (SPs), which are temporary structures on the N-termini of their nascent chains. During translation, such proteins enter the lumen and membrane of the ER by a process known as co-translational translocation. Small molecules have been found that interfere with this process, decreasing protein expression by recognizing the unique structures of the SPs of particular proteins. The SP may thus become a validated target for designing drugs for numerous disorders, including certain hereditary diseases.

Syntheses and anti-HIV and human cluster of differentiation 4 (CD4) down-modulating potencies of pyridine-fused cyclotriazadisulfonamide (CADA) compounds.

CADA compounds selectively down-modulate human cell-surface CD4 protein and are of interest as HIV entry inhibitors and as drugs for asthma, rheumatoid arthritis, diabetes and some cancers. Postulating that fusing a pyridine ring bearing hydrophobic substituents into the macrocyclic scaffold of CADA compounds may lead to potent compounds with improved properties, 17 macrocycles were synthesized, 14 with 12-membered rings having an isobutylene head group, two arenesulfonyl side arms, and fused pyridine rings bearing a para substituent. The analogs display a wide range of CD4 down-modulating and anti-HIV potencies, including some with greater potency than CADA, proving that a highly basic nitrogen atom in the 12-membered ring is not required for potency and that hydrophobic substituents enhance potency of pyridine-fused CADA compounds. Cytotoxicities of the new compounds compared favorably with those of CADA, showing that incorporation of a pyridine ring into the macrocyclic scaffold can produce selective compounds for potently down-modulating proteins of medicinal interest.

A Proteomic Survey Indicates Sortilin as a Secondary Substrate of the ER Translocation Inhibitor Cyclotriazadisulfonamide (CADA).

The small molecule CADA was shown to down-modulate the expression of human CD4 in a signal peptide-dependent way through inhibition of its cotranslational translocation across the ER membrane. Previous studies characterizing general glycoprotein levels and the expression of 14 different cell surface receptors showed selectivity of CADA for human CD4. Here, a PowerBlot Western Array was used as a screen to analyze the proteome of CADA-treated SUP-T1 human CD4+ T lymphocytes. This high-throughput monoclonal antibody panel-based immunoblotting assay of cellular signaling proteins revealed that only a small subset of the 444 detected proteins was differentially expressed after treatment with CADA. Validation of these proteomic data with optimized immunoblot analysis confirmed the CADA-induced change in expression of the cell cycle progression regulator pRb2 and the transcription factor c-Jun. However, the up-regulation of pRb2 or down-modulation of c-Jun by CADA had no impact on cell cycle transition. Also, the reduced protein level of human CD4 did not inhibit T cell receptor signaling. Interestingly, the signal peptide-containing membrane protein sortilin was identified as a new substrate for CADA. Both cellular expression and in vitro cotranslational translocation of sortilin were significantly reduced by CADA, although to a lesser extent as compared with human CD4. Our data demonstrate that a small signal peptide-binding drug is able to down-modulate the expression of human CD4 and sortilin, apparently with low impact on the cellular proteome.

Proton-Gated Photoisomerization of Amino-Substituted Dibenzofulvene Rotors.

Derivatives of 9-(2,2,2-triphenylethylidene)-fluorene (TEF) undergo E/Z photoisomerization and are candidates for light-powered molecular actuators and switches. The 2-substituted derivatives bearing an amino group (ATEF) or a dimethylamino group (DTEF) are weakly photoactive in the absence of acids, but protonation increases photoisomerization quantum yields by factors of 30-60. Such compounds may be useful for incorporation into pH-switchable photoactive devices.

Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation.

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.

Synthesis and Photoisomerization of Substituted Dibenzofulvene Molecular Rotors.

The synthesis, spectral and structural characterization, and photoisomerization of a family of 2-substituted dibenzofulvene molecular actuators based on (2,2,2-triphenylethylidene)fluorene (TEF) are reported. The 2-substituted species investigated are nitro (NTEF), cyano (CTEF), and iodo (ITEF). X-ray structures of these three compounds and three intermediates were determined to assign alkene configuration and investigate the effects of the 2-substituents on steric gearing. The addition-elimination reaction of Z-9 with trityl anion to form Z-10 proceeded with complete retention of configuration. Rates of photoisomerization were measured at irradiation wavelengths between 266-355 nm in acetonitrile/dioxane solutions at room temperature. Photoisomerization quantum yields (φ) were calculated by means of a mathematical model that accounts for a certain degree of photodecomposition in the cases of CTEF and ITEF. Quantum yields vary significantly with substituent, having maximum values of φ=0.26 for NTEF, 0.39 for CTEF, and 0.50 for ITEF. NTEF is photochemically robust and has a large quantum yield for photoisomerization in the near-UV, making it a particularly promising drive rotor moiety for light-powered molecular devices.

Metal complexes of pyridine-fused macrocyclic polyamines targeting the chemokine receptor CXCR4.

The chemokine receptor CXCR4 acts as a key cell surface receptor in HIV infections, multiple forms of cancer, and various other pathologies, such as rheumatoid arthritis and asthma. Macrocyclic polyamines and their metal complexes are known to exert anti-HIV activity, many acting as HIV entry inhibitors by specifically binding to CXCR4. Three series of pyridopentaazacylopentadecanes, in which the pyridine ring is fused to zero, one, or two saturated six-membered rings, were synthesized by manganese(ii)-templated Schiff-base cyclization of triethylenetetramine with various dicarbonyl compounds. By evaluating these macrocyclic polyamines and their complexes with Mn(2+), Cu(2+), Fe(3+), and Zn(2+), we have discovered novel CXCR4-binding compounds. The MnCl2 complex of a new pentaazacyclopentadecane with one fused carbocyclic ring (11) was found to have the greatest potency as an antagonist of the chemokine receptor CXCR4 (IC50: 0.014 µM), as evidenced by inhibiting binding of CXCL12 to PBMCs (peripheral blood mononuclear cells). Consequently, this compound inhibits replication of the CXCR4-using (X4) HIV-1 strain NL4-3 in the TZM-bl cell line with an IC50 value of 0.52 µM and low cytotoxicity (CC50: >100 µM). In addition, 18 other compounds were evaluated for their interaction with CXCR4 via their ability to interfere with ligand chemokine binding and HIV entry and infection. Of these, the metal complexes of the two more hydrophobic series with one or two fused carbocyclic rings exhibited the greatest potency. The Zn(2+) complex 21 was among the most potent, showing that redox activity of the metal center is not associated with CXCR4 antagonist activity.

Comparison of in vitro membrane permeabilities of diverse environmental chemicals with in silico predictions.

The parallel artificial membrane permeability assay (PAMPA) is widely used for estimating biomembrane permeabilities of experimental drugs in pharmaceutical research. However, there are few reports of studies using PAMPA to measure membrane permeabilities of chemicals of environmental concern (CECs) outside the pharmaceutical domain, many of which differ substantially from drugs in their physicochemical properties. We applied PAMPA methods simulating gastrointestinal (PAMPA-GIT) and blood-brain barrier (PAMPA-BBB) membranes under consistent conditions to 51 CECs, including some pharmaceuticals. A backward stepwise multivariate linear regression was implemented to explore the correlation between the differences of measured permeabilities from PAMPA-GIT and PAMPA-BBB and Abraham solute descriptors. In addition, a previously reported in silico model was evaluated by comparing predicted and measured permeability results. PAMPA-GIT and PAMPA-BBB experimental permeability results agreed relatively well. The backward stepwise multivariate linear regression identified excess molar refraction and polarizability to be significant at the 0.10 level in predicting the differences between PAMPA-GIT and PAMPA-BBB. The in silico model performed well - with predicted permeability of most compounds within two-fold of experimentally measured values. We found that CECs pose experimental challenges to the PAMPA method in terms of having lower solubility and lower stability compared to most drugs.

Role of Sec61α2 translocon in insulin biosynthesis.

Translocational regulation of proinsulin biosynthesis in pancreatic ß-cells is unknown, although several studies have reported an important accessory role for the Translocon-Associated Protein complex to assist preproinsulin delivery into the endoplasmic reticulum via the heterotrimeric Sec61 translocon (comprised of α, ß, and γ subunits). The actual protein-conducting channel is the α-subunit encoded either by Sec61A1 or its paralog Sec61A2. Although the underlying channel selectivity for preproinsulin translocation is unknown, almost all studies of Sec61α to date have focused on Sec61α1. There is currently no evidence to suggest that this gene product plays a major role in proinsulin production, whereas genome-wide association studies indicate linkage of Sec61A2 with diabetes. Here, we report that evolutionary differences in mouse preproinsulin signal peptides affect proinsulin biosynthesis. Moreover, we find that although some preproinsulin translocation can proceed through Sec61α1, Sec61α2 has a greater impact on proinsulin biosynthesis in pancreatic ß-cells. Remarkably, Sec61α2-translocon deficiency exerts a significant inhibitory effect on the biosynthesis of preproinsulin itself, including a disproportionate increase of full-length nacent chain unreleased from ribosomes. This study not only reveals novel translocational regulation of proinsulin biosynthesis, but also provides a rationale for genetic evidence suggesting an important role of Sec61α2 in maintaining blood glucose homeostasis.

Isoform selectivities of novel 4-hydroxycoumarin imines as inhibitors of myosin II.

Muscle myosin inhibition could be used to treat many medical conditions involving hypercontractile states, including muscle spasticity, chronic musculoskeletal pain, and hypertrophic cardiomyopathy. A series of 13 advanced analogs of 3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one (BHC) were synthesized to explore extended imine nitrogen side chains and compare aldimines vs. ketimines. None of the new analogs inhibit nonmuscle myosin in a cytokinesis assay. ATPase structure-activity relationships reveal that selectivity for cardiac vs. skeletal myosin can be tuned with subtle structural changes. None of the compounds inhibited smooth muscle myosin II. Docking the compounds to homology models of cardiac and skeletal myosin II gave rationales for the effects of side arm length on inhibition selectivity and for cardiac vs. skeletal myosin. Properties including solubility, stability and toxicity, suggest that certain BHC analogs may be useful as candidates for preclinical studies or as lead compounds for advanced candidates for drugs with cardiac or skeletal muscle myosin selectivity.

Controlling the spatial orientation of molecular actuators: polarized photoisomerization of 2-nitro-9-(2,2,2-triphenylethylidene)fluorene in a thin polymer matrix.

The molecule 2-nitro-9-(2,2,2-triphenylethylidene)fluorene (NTEF) was studied as a potential light-driven molecular motor. Absorption at 355 nm causes a reversible spatial reorientation of the angular distribution of the dibenzofulvene rotor moiety of NTEF when immobilized in a poly(methyl methacrylate) (PMMA) matrix adsorbed on a fused silica surface in air at room temperature. The photoreorientation dynamics was probed by polarized normal incidence cavity ringdown spectroscopy (NICRDS) when the matrix was irradiated by linearly polarized "drive" light. Polarized drive irradiation at 355 nm creates a "hole" in the angular distribution of the molecular transition dipoles. Changing the polarization of the drive beam refills the hole, creating a new hole. A stochastic model was fitted to the experimental hole burning measurements to obtain a photoreorientation quantum yield (Φ(reorient) = 0.014). The photoreorientation process appears to be driven by photoisomerization of the exocyclic dibenzofulvene double bond of NTEF.

Reduction of Progranulin-Induced Breast Cancer Stem Cell Propagation by Sortilin-Targeting Cyclotriazadisulfonamide (CADA) Compounds.

Cyclotriazadisulfonamide (CADA) compounds selectively down-modulate two human proteins of potential therapeutic interest, cluster of differentiation 4 (CD4) and sortilin. Progranulin is secreted from some breast cancer cells, causing dedifferentiation of receiving cancer cells and cancer stem cell proliferation. Inhibition of progranulin binding to sortilin, its main receptor, can block progranulin-induced metastatic breast cancer using a triple-negative in vivo xenograft model. In the current study, seven CADA compounds (CADA, VGD020, VGD071, TL020, TL023, LAL014, and DJ010) were examined for reduction of cellular sortilin expression and progranulin-induced breast cancer stem cell propagation. In addition, inhibition of progranulin-induced mammosphere formation was examined and found to be most significant for TL020, TL023, VGD071, and LAL014. Full experimental details are given for the synthesis and characterization of the four new compounds (TL020, TL023, VGD071, and DJ010). Comparison of solubilities, potencies, and cytotoxicities identified VGD071 as a promising candidate for future studies using mouse breast cancer models.

Human immunodeficiency virus type 1 escape from cyclotriazadisulfonamide-induced CD4-targeted entry inhibition is associated with increased neutralizing antibody susceptibility.

Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients.

Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization.

BACKGROUND: HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. RESULTS: We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). CONCLUSION: These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa.

Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.

Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo, and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC50) <1 µM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.

Tsuji-Trost Cyclization of Disulfonamides: Synthesis of 12-Membered, 11-Membered, and Pyridine-Fused Macrocyclic Triamines.

Macrocyclic triamine disulfonamides can be synthesized by double Tsuji-Trost N-allylation reaction of open-chain disulfonamides with 2-alkylidene-1,3-propanediyl bis(carbonates). The previously used Atkins-Richman macrocyclization method generally gives lower yields and requires more tedious purification of the product. Solvent, palladium source, ligand, and concentration have all been varied to optimize the yields of two key 12-membered ring bioactive compounds, CADA and VGD020. The new approach tolerates a wide range of functional groups and gives highest yields for symmetrical compounds in which the acidities of the two sulfonamide groups are matched, although the yields of unsymmetrical compounds are still generally good. The method has also been extended to the synthesis of 11-membered rings, pyridine-fused macrocycles, and products bearing an ester or aryl substituent on the exocyclic double bond.

Design and cellular kinetics of dansyl-labeled CADA derivatives with anti-HIV and CD4 receptor down-modulating activity.

A new class of anti-retrovirals, cyclotriazadisulfonamide (CADA) and its derivatives, specifically down-regulate CD4, the main receptor of HIV, and prevent HIV infection in vitro. In this work, several CADA derivatives, chemically labeled with a fluorescent dansyl group, were evaluated for their biological features and cellular uptake kinetics. We identified a derivative KKD-016 with antiviral and CD4 down-modulating capabilities similar to those of the parental compound CADA. By using flow cytometry, we demonstrated that the dose-dependent cellular uptake of this derivative correlated with CD4 down-modulation. The uptake and activity of the dansyl-labeled compounds were not dependent on the level of expression of CD4 at the cell surface. Removal of the CADA compounds from the cell culture medium resulted in their release from the cells followed by a complete restoration of CD4 expression. The inability of several fluorescent CADA derivatives to down-modulate CD4 was not associated with their lower cellular uptake and was not reversed by facilitating their cell penetration by a surfactant. These results prove the successful integration of the dansyl fluorophore into the chemical structure of a CD4 down-modulating anti-HIV compound, and show the feasibility of tracking a receptor and its down-modulator simultaneously. These fluorescent CADA analogs with reversible CD4 down-regulating potency can now be applied in further studies on receptor modulation, and in the exploration of their potentials as preventive and therapeutic anti-HIV drugs.

Inhibitors of HIV infection via the cellular CD4 receptor.

Recent advances in our understanding of cellular and molecular mechanisms of viral penetration of the target cell have provided the basis for novel chemotherapy and prophylaxis of HIV-1 infections. This knowledge has been successfully applied to the development of inhibitors that target discrete steps of the entry process. Interesting approaches for prevention of HIV-1 entry include the use of small-molecule inhibitors, natural ligands and/or monoclonal antibodies that interfere with gp120/CD4 interaction. Other compounds acting by novel mechanisms have recently been identified as anti-HIV agents and seem worthy of further preclinical development. Of particular interest in this regard are cyclotriazadisulfonamide (CADA) compounds, which down-modulate the cellular receptor, CD4. A series of analogues of 9-benzyl-3-methylene-1,5-di-p-toluenesulfonyl-1,5,9-triazacyclododecane (CADA) has been synthesized and tested for CD4 down-modulation and anti-HIV activity. Some derivatives proved to be highly effective in decreasing cellular CD4 and in acting as HIV entry inhibitors. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies correlating molecular features with potency have been used to produce a computational model. This model can be used to design more potent CD4 down-modulating drugs for HIV therapy and prophylaxis. This review summarizes the results of recent studies relating to inhibitors of HIV infection via CD4 and discusses the therapeutic potential of targeting this cellular receptor. Special attention is given to our own work on small-molecule HIV entry inhibitors endowed with CD4 down-modulating properties.