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The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = −0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide–antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitroactivity (hNaV1.7 IC50= 250 nM) and without in vivoactivity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide–antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitropotency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivowith accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaVin vitroactivity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide–antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivohalf-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = −0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies.SIGNIFICANCE STATEMENTAlthough multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaVthat are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaVsubtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes and that altering these residues can have dramatic effects on NaVinhibitory activity. We demonstrate that strong peptide–membrane interactions are not essential for inhibiting NaV1.7 and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaVresidues, thereby affecting peptide−channel interactions. Our detailed structure–activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.

Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivoproperties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm= 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm> 95 °C), high affinity for VEGF-A (Kd= 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivoin mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

Hepcidin is a four disulfide 25residue peptide hormone which has a central role in the regulation of iron homeostasis. To support studies on hepcidin we have sought to establish reliable and robust synthetic methods for the preparation of correctly folded materials. While correctlyfolded hepcidin has good aqueous solubility, we have found that its direct synthetic precursor, linear reduced hepcidin peptide, is resistant to solubilization, prone to precipitation at pH ≥ 6, and thus difficult to fold efficiently. Attempts to directly fold either the crude or purified linear hepcidin peptide by air or DMSO oxidation methods under basic conditions were ineffective. However, addition of a glutathione redox pair system improved folding of purified linear hepcidin at mild basic pH pH 7.5. Under acidic conditions, it was possible to oxidatively fold both crude and purified hepcidin using a polymersupported oxidizing strategy. Peptide precipitation was also avoided under acidic conditions. Isolated folding yields of human hepcidin under acidic polymerassisted conditions were superior to yields under basic folding conditions. These studies enabled identification of a reliable synthetic route for correctlyfolded hepcidin. © 2010 Wiley Periodicals, Inc. Biopolymers Pept Sci 94: 257–264, 2010.

The efficient solid-phase glycosylation of amino acid side chains (serine (Ser), threonine (Thr), and tyrosine (Tyr)) in peptides was demonstrated with a variety of glycosyl trichloroacetimidate donors in high yields and purities. A novel photolabile linker, with no chiral centre, was introduced to facilitate analysis by both matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and nanoprobe magic angle spinning (MAS) NMR spectroscopy. Product analysis by nanoprobe MAS NMR spectroscopy, LC-MS and MALDI-TOF mass spectrometry of the glycosylation reactions indicated that the reactivity order of the hydroxy side-chain functions of amino acids in peptides on the solid-phase was Tyr>Ser>Thr. The nearly quantitative glycosylation yields and the efficient on-bead product analysis by nanoprobe MAS NMR spectroscopy have made a truly solid-phase approach for the synthesis and analysis of glycopeptide libraries possible. Supporting information for this article is available on the WWW under http://wiley-vch.de/home/chemistry/ or from the author.

The efficient solid‐phase glycosylation of amino acid side chains (serine (Ser), threonine (Thr), and tyrosine (Tyr)) in peptides was demonstrated with a variety of glycosyl trichloroacetimidate donors in high yields and purities. A novel photolabile linker, with no chiral centre, was introduced to facilitate analysis by both matrix‐assisted laser desorption ionisation time of flight (MALDI‐TOF) mass spectrometry and nanoprobe magic angle spinning (MAS) NMR spectroscopy. Product analysis by nanoprobe MAS NMR spectroscopy, LC‐MS and MALDI‐TOF mass spectrometry of the glycosylation reactions indicated that the reactivity order of the hydroxy side‐chain functions of amino acids in peptides on the solid‐phase was Tyr>Ser>Thr. The nearly quantitative glycosylation yields and the efficient on‐bead product analysis by nanoprobe MAS NMR spectroscopy have made a truly solid‐phase approach for the synthesis and analysis of glycopeptide libraries possible.

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)‐binding protein), a new member of the S100 family of EF‐hand calcium‐binding proteins, was chemically synthesised using highly optimised 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/tert‐butoxycarbonyl in situ neutralisation solid‐phase chemistry. Circular dichroism studies indicated that CaCl2decreased the helical content by 27% whereas helicity was marginally increased by ZnCl2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time‐of‐flight mass spectrometry which clearly demonstrated the prevalence of the non‐covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/ tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl 2decreased the helical content by 27% whereas helicity was marginally increased by ZnCl 2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

The Human Genome Project and other major sequencing projects have rapidly provided a vast array of new protein sequences. In the postgenomic era, the physical form of many of these gene‐encoded sequences will be vital for biomedical research and drug development. In this epoch, the advantages of protein chemical synthesis will complement recombinant‐DNA methods, and will be used to provide rapid access to small proteins or functional receptor domains. In this review the key methodological advances that have made the synthesis of long peptides and small proteins more effective will be presented. Focus is given to the issues and goals of contemporary chemical protein synthesis, including (1) the rapid chain assembly of tailored peptide segments for use in ligation strategies, and (2) development of highly efficient and universal chemoselective ligation strategies.© 2000 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 55: 217–226, 2000

The Human Genome Project and other major sequencing projects have rapidly provided a vast array of new protein sequences. In the postgenomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. In this epoch, the advantages of protein chemical synthesis will complement recombinant-DNA methods, and will be used to provide rapid access to small proteins or functional receptor domains. In this review the key methodological advances that have made the synthesis of long peptides and small proteins more effective will be presented. Focus is given to the issues and goals of contemporary chemical protein synthesis, including (1) the rapid chain assembly of tailored peptide segments for use in ligation strategies, and (2) development of highly efficient and universal chemoselective ligation strategies.© 2000 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 55: 217–226, 2000

Inhibition of the pituitary adenylate cyclase 1 receptor (PAC1R) is a novel mechanism that could be used for abortive treatment of acute migraine. Our research began with comparative analysis of known PAC1R ligand scaffolds, PACAP38 and Maxadilan, which resulted in the selection of des(24–42) Maxadilan, 6, as a starting point. C-terminal modifications of 6improved the peptide metabolic stability in vitroand in vivo. SAR investigations identified synergistic combinations of amino acid replacements that significantly increased the in vitroPAC1R inhibitory activity of the analogs to the pM IC90range. Our modifications further enabled deletion of up to six residues without impacting potency, thus improving peptide ligand binding efficiency. Analogs 17and 18exhibited robust in vivoefficacy in the rat Maxadilan-induced increase in blood flow (MIIBF) pharmacodynamic model at 0.3 mg/kg subcutaneous dosing. The first cocrystal structure of a PAC1R antagonist peptide (18) with PAC1R extracellular domain is reported.

We report a metal free synthetic hydrogel copolymer with affinity and selectivity for His6-tagged peptides and proteins. Small libraries of copolymers incorporating charged and hydrophobic functional groups were screened by an iterative process for His6 peptide affinity. The monomer selection was guided by interactions found in the crystal structure of an anti-His tag antibody-His6 peptide antigen complex. Synthetic copolymers incorporating a phenylalanine-derived monomer were found to exhibit strong affinity for both His6-containing peptides and proteins. The proximity of both aromatic and negatively charged functional groups were important factors for the His6 affinity of hydrogel copolymers. His6 affinity was not compromised by the presence of enzyme cleavage sequences. The His6–copolymer interactions are pH sensitive: the copolymer selectively captured His6 peptides at pH 7.8 while the interactions were substantially weakened at pH 8.6. This provided mild conditions for releasing His6-tagged proteins from the copolymer. Finally, a synthetic copolymer coated chromatographic medium was prepared and applied to the purification of a His6-tagged protein from an E. coliexpression system. The results establish that a synthetic copolymer-based affinity medium can function as an effective alternative to immobilized metal ion columns for the purification of His6-tagged proteins.

No AbstractSupporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2001/z17269_s.pdfor from the author.

No AbstractSupporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2001/z17269_s.pdf or from the author.

No Abstract Supporting information for this article is available on the WWW under http://www.angewandte.com or from the author.

For Abstract see ChemInform Abstract in Full Text.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The cover picture showsa new concept in protecting-group chemistry termed unichemo protection (UCP). This strategy only requires a single chemical process for all deprotection reactions. The UCP protecting groups are derived from a repetitive unit that permits their controlled and efficient step-wise removal. Functional-site selectivity is achieved by varying the degree of oligomerization at each site, and, after each deprotection cycle, only the newly liberated functional site is available for derivatization. The UCP strategy does not impose a restriction on the possible number of selectively protected sites in a molecule. The low-energy conformer of the pentalysine scaffold assembled with N-sec-butylglycyl protecting-group units is shown bottom left. By using the UCP approach the five protecting groups were sequentially removed and the exposed amino acid groups functionalized with five different organic acids. UCP facilitates an orthogonal process that is not dependent on a range of finely tuned and differently compatible processes. Moreover, since UCP is based on uniform deprotection reactions, the requirement of reaction compatibility with other parts of a molecule only increases linearly with the degree of polyfunctionalization (graph, bottom right). That is, after the initial requirement of parent-molecule stability is satisfied, only the sequential requirements towards each newly introduced group is an issue. In contrast, a quadratic increase in complexity with respect to the number of protected functional groups, even in the simplest cases, accompanies existing orthogonal protection strategies. More about the UCP strategy is reported by L. P. Miranda and M. Meldal on p. 3655ff.

Das Titelbild zeigt ein neues Konzept in der Schutzgruppenchemie – die Unichemo-Schutzgruppenstrategie (unichemo protection, UCP). Ein einziger chemischer Prozess reicht hierbei für alle Entschützungsreaktionen aus. Bei der UCP werden als Schutzgruppen Oligomere aus Wiederholungseinheiten verwendet, sie können in kontrollierter und effizienter Weise Schritt für Schritt entfernt werden. Selektivität für eine bestimmte funktionalisierte Stelle in einem Molekül wird durch unterschiedlichen Oligomerisierungsgrad erreicht, und nach jedem Entschützungsschritt ist nur die neu freigesetzte Gruppe für weitere Derivatisierung verfügbar. Bei der UCP-Strategie gibt es keine Obergrenze für die Zahl selektiv geschützter Stellen. Unten links ist das Konformer des Pentalysin-Gerüsts mit der niedrigsten Energie, ausgestattet mit N-sec-Butylglycyl-Schutzgruppeneinheiten, gezeigt. Fünf Schutzgruppen wurden nacheinander entfernt und die jeweils freigesetzten Gruppen mit fünf verschiedenen organischen Säuren derivatisiert. Durch UCP kann eine orthogonale Schutzgruppenstrategie vereinfacht werden, da man sich hierbei nicht einer Reihe sorgfältig aufeinander abgestimmter Reaktionen bedienen muss. Ferner nimmt die Notwendigkeit der Reaktionskompatibilität mit anderen Teilen eines Moleküls nur linear mit dem Polyfunktionalisierungsgrad zu (siehe Graph unten rechts) – selbst im einfachsten Fall besteht bei üblichen orthogonalen Schutzgruppenstrategien eine quadratische Abhängigkeit. Ausführlich werden Sie über die UCP-Strategie informiert in der Zuschrift von L. P. Miranda und M. Meldal auf S. 3767ff.

The cover picture shows a new concept in protecting-group chemistry termed unichemo protection (UCP). This strategy only requires a single chemical process for all deprotection reactions. The UCP protecting groups are derived from a repetitive unit that permits their controlled and efficient step-wise removal. Functional-site selectivity is achieved by varying the degree of oligomerization at each site, and, after each deprotection cycle, only the newly liberated functional site is available for derivatization. The UCP strategy does not impose a restriction on the possible number of selectively protected sites in a molecule. The low-energy conformer of the pentalysine scaffold assembled with N-sec-butylglycyl protecting-group units is shown bottom left. By using the UCP approach the five protecting groups were sequentially removed and the exposed amino acid groups functionalized with five different organic acids. UCP facilitates an orthogonal process that is not dependent on a range of finely tuned and differently compatible processes. Moreover, since UCP is based on uniform deprotection reactions, the requirement of reaction compatibility with other parts of a molecule only increases linearly with the degree of polyfunctionalization (graph, bottom right). That is, after the initial requirement of parent-molecule stability is satisfied, only the sequential requirements towards each newly introduced group is an issue. In contrast, a quadratic increase in complexity with respect to the number of protected functional groups, even in the simplest cases, accompanies existing orthogonal protection strategies. More about the UCP strategy is reported by L. P. Miranda and M. Meldal on p. 3655ff.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.