A covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the HIV-1 envelope complex

Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.     

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.     

Development of a Ga-68 labeled PET tracer with short linker for prostate-specific membrane antigen (PSMA) targeting

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with ⁶⁸Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with ⁶⁸Ga using generator-eluted ⁶⁸GaCl₃ in 0.1?M HCl in the presence of 1?M NaOAc at pH 5.5. The stabilities of ⁶⁸Ga-labeled compounds in human serum were tested at 37.5?°C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [¹²⁵I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC₅₀ of Ga-NOTA-GUL was 18.3?nM. In the biodistribution study, tumor uptake of ⁶⁸Ga-NOTA-GUL (5.40% ID/g) was higher than that of ⁶⁸Ga-DOTA-GUL (4.66% ID/g) at 1?h. Tumor/muscle and tumor/blood uptake ratios of ⁶⁸Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of ⁶⁸Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of ⁶⁸Ga-NOTA-GUL was 3.4-fold higher than that of ⁶⁸Ga-DOTA-GUL. ⁶⁸Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized ⁶⁸Ga-NOTA-GUL and ⁶⁸Ga-DOTA-GUL for prostate cancer imaging. ⁶⁸Ga-NOTA-GUL showed better radiochemical and biodistribution results. ⁶⁸Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical.     

The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.     

Prodrug and covalent linker strategies for the solubilization of dual-action antioxidants/iron chelators

Water soluble prodrugs of hybrid free radical scavenger/iron chelating molecules, based on 3,5-disubstituted-4-hydroxyphenyl derivatives and 3-hydroxy-2-methyl-4(1H)-pyridinone (deferiprone), have been prepared. Related hybrid molecules containing a covalent poly(ethylene)glycol or an amine linker were also synthesized.     

Functionalized membrane supports for covalent protein microsequence analysis

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).     

Effects of linker length and flexibility on multivalent targeting

Increasing valence can enhance the ability of molecular targeting constructs to bind specifically to targeted cells for drug delivery. Here, we mathematically model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approximate varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce experimentally measured avidities. Also, the magnitude of VR, in conjunction with receptor density and ligand concentration, significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging.     

Activation of a covalent outer membrane phospholipase A dimer.

The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.     

Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 A resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36 and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute that connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid-binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex.     

A novel Golgi membrane protein is part of a GTPase-binding protein complex involved in vesicle targeting

Through two-hybrid interactions, protein affinity and localization studies, we previously identified Yip1p, an integral yeast Golgi membrane protein able to bind the Ras-like GTPases Ypt1p and Ypt31p in their GDP-bound conformation. In a further two-hybrid screen, we identified Yif1p as an interacting factor of Yip1p. We show that Yif1p is an evolutionarily conserved, essential 35.5 kDa transmembrane protein that forms a tight complex with Yip1p on Golgi membranes. The hydrophilic N-terminal half of Yif1p faces the cytosol, and according to two-hybrid analyses can interact with the transport GTPases Ypt1p, Ypt31p and Sec4p, but in contrast to Yip1p, this interaction is dispensable for Yif1 protein function. Loss of Yif1p function in conditional-lethal mutants results in a block of endoplasmic reticulum (ER)-to-Golgi protein transport and in an accumulation of ER membranes and 40-50 nm vesicles. Genetic analyses suggest that Yif1p acts downstream of Yip1p. It is inferred that Ypt GTPase binding to the Yip1p-Yif1p complex is essential for and precedes vesicle docking and fusion.     

Signal sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence-independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin

The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin- glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues.     

An advanced affinity membrane for covalent binding of amino ligands

We report here an advanced, chemically active and yet hydrolytically stable microporous membrane which allows permanent covalent binding of amino ligands such as proteins. Rapid, single-step immobilizations produce a high density of immobilized ligands. Surface chemistry of the membrane is specifically designed to have extremely low nonspecific binding. Binding characteristics of the UltraBind membrane, various immobilization techniques and optimum immobilization conditions for diagnostic immunoassays are described.     

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA₁ displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA₁ derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA₁ (PGA₁-biotinamide, PGA₁-B), to establish its validity to identify cyPG–protein interactions of potential therapeutic interest. PGA₁ and PGA₁-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA₁-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA₁-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA₁-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA₁ that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.     

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)). EII(mtl) overexpression was under control of the lambdaP(R) promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the lambdaP(R) promoter. Together with the presence of the repressor gene on the EII(mtl) plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EII(mtl) samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting.

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.