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The number of new toxin peptide discoveries has been rapidly growing in the past few decades. Because of progress in proteomics, sequencing technologies, and high throughput bioassays, the search for new toxin peptides from venom collections and potency optimization has become manageable. However, to date, only six toxin peptide-derived therapeutics have been approved by the USFDA, with only one, ziconotide, for a pain indication. The challenge of venom-derived peptide therapeutic development remains in improving selectivity to the target and more importantly, in delivery of these peptides to the sites of action in the central and peripheral nervous system. In this review, we highlight peptide toxins that target major therapeutic targets for pain and discuss the challenges of developing toxin peptides as potential therapeutics. [ABSTRACT FROM AUTHOR]

Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site- specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL- 5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-H IV therapeutics which are still desperately needed. [ABSTRACT FROM AUTHOR]

Examines the efficacy of using activated N[sup alpha]-acyl transfer auxiliary for amide-backbone substitution of hindered peptides. Primary function of the activated auxiliary; Incorporation of Hnb auxiliary at the N[sup alpha]-amine; Implication of Hnb-assisted Fmoc solid-phase synthesis STAT-91 for the coupling efficiencies of hindered peptides.

Presents information on a study which analyzed the accelerated chemical synthesis of peptides and small proteins. Experimental procedures; Results and discussion.

Presents a study defining the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF (a highly volatile, nonoxidizing acid with a strong protonating ability) cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. What is one of the main challenges in peptide synthesis; Identification of the most effective and commonly used acid for treating the resin bound peptides; Results and discussion of the study.

One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1,2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats. [Display omitted] • Nanoparticle vaccine comprised of two subunits: Component A and Component B. • Generation of FIH material of Component A in support of clinical development. • Development and GMP manufacturing on an extremely accelerated timeline of 6 months. • Leveraged stable pool instead of clone reduces development timeline by 2–3 months. • A potential process platform to use across different Component A molecules. [ABSTRACT FROM AUTHOR]

The advent of recombinant protein-based therapeutic agents in the 1980s and subsequent waves of innovation in molecular biology and engineering of biologics has permitted the production of an increasingly broad array of complex, high molecular weight constructs. While this has opened a powerful new toolbox of molecular scaffolds with which to probe and interdict biological processes, it also makes deciphering the architectural nuances between individual constructs intuitively difficult. Key to downstream data processes for the detection of data trends is the ability to unambiguously identify, compare, and communicate the nature of molecular compositions. Existing small molecule orientated software tools are not intended for structures such as peptides, proteins, antibodies, and RNA, and do not contain adequate atomistic or domain-level detail to appropriately convey their higher structural complexity. Similarly, there is a paucity of large molecule-focused data analysis and visualization tools. This article will describe four new approaches we developed for the graphical representation and analysis of complex large molecules and experimental data. These tools help fulfill key needs in scientific communication and structure-property analysis of complex biologics and modified oligonucleotide-based drug candidates. [ABSTRACT FROM AUTHOR]

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers. [ABSTRACT FROM AUTHOR]

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work. [ABSTRACT FROM AUTHOR]

This article presents a study related to chemical synthesis of keto-proteins which was conducted as of November 26, 2003. Keto-peptides are important derivatives for participating in chemoselective conjugation processes with aminooxy compounds in aqueous solution. The introduction of keto groups into peptides in the form of levulinic, pyruvic or keto-containing amino acids can be carried out during or after solid-phase chain assembly at an orthogonally protected amine, such as the amino group of a given lysine residue. Transamination of N-terminal peptide residues with sodium glyoxylate and copper catalysis is another route to keto-peptides.

Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel Na V 1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the Na V 1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to Na V 1.7, we have mapped the binding site to transmembrane segments 1–4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using Na V 1.5/Na V 1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting Na V 1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules. [ABSTRACT FROM AUTHOR]

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp29, Lys31, and Phe34 near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5. [ABSTRACT FROM AUTHOR]

We describe a novel epitope discovery strategy for creating an affinity agent/peptide tag pair. A synthetic polymer nanoparticle (NP) was used as the "bait" to catch an affinity peptide tag. Biotinylated peptide tag candidates of varied sequence and length were attached to an avidin platform and screened for affinity against the polymer NP. NP affinity for the avidin/peptide tag complexes was used to provide insight into factors that contribute NP/tag binding. The identified epitope sequence with an optimized length (tMel-tag) was fused to two recombinant proteins. The tagged proteins exhibited higher NP affinity than proteins without tags. The results establish that a fusion peptide tag consisting of optimized 15 amino acid residues can provide strong affinity to an abiotic polymer NP. The affinity and selectivity of NP/tMel-tag interactions were exploited for protein purification in conjunction with immobilized metal ion/His6-tag interactions to prepare highly purified recombinant proteins. This strategy makes available inexpensive, abiotic synthetic polymers as affinity agents for peptide tags and provides alternatives for important applications where more costly affinity agents are used. [ABSTRACT FROM AUTHOR]

Review: 26 refs. [ABSTRACT FROM AUTHOR]

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys1-Cys8, Cys3-Cys6, Cys2-Cys4, and Cys5-Cys7. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia. Methods: An LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays. Results: The calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at −60 to −80 °C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were −11.2% and 5.6%, respectively at the LLOQ, and less than ±7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between −0.7 and +2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n =16, median 121 ng/mL) and 99 ng/mL (n =50, median 68 ng/mL), respectively. Conclusions: A fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples. [Copyright &y& Elsevier]

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure–function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family. [Copyright &y& Elsevier]

We report the design and total chemical synthesis of “synthetic erythropoiesis protein” (SEP), a 51-kitodattort protein-polymer constryct consisting of a 166-amino-acid potypeptide chain and two covatently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalerrt structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 ±10 dallons by electrospray mass spectromctry, and with a pl of 5.0. In celt and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

• Lipidated apelin-13 showed in an increase in ejection fraction in an anesthetized rat myocardial infarction model. • Rat plasma protein binding of C13-lipidated apelin-13 was measured. • Rat PK (iv and sc) was measured for 15-mer peptide APJ agonists; half-life extension was observed for these peptides. • The length and type (fatty acid or lipid diacid) of lipid group affects the absorptive phase of subcutaneous dosing. • Lipidation increases plasma protein binding and extends the half-lives of peptides. Agonism of the endothelial receptor APJ (putative receptor protein related to AT 1 ; AT 1 : angiotensin II receptor type 1) has the potential to ameliorate congestive heart failure by increasing cardiac output without inducing hypertrophy. Although the endogenous agonist, pyr-apelin-13 (1), has shown beneficial APJ-mediated inotropic effects in rats and humans, such effects are short-lived given its extremely short half-life. Here, we report the conjugation of 1 to a fatty acid, providing a lipidated peptide (2) with increased stability that retains inotropic activity in an anesthetized rat myocardial infarction (MI) model. We also report the preparation of a library of 15-mer APJ agonist peptide-lipid conjugates, including adipoyl-γGlu-OEG-OEG-hArg-r-Q-hArg-P-r-NMeLeu S H K-G-Oic-pIPhe-P-DBip-OH (17), a potent APJ agonist with high plasma protein binding and a half-life suitable for once-daily subcutaneous dosing in rats. A correlation between subcutaneous absorption rate and lipid length/type of these conjugates is also reported. [ABSTRACT FROM AUTHOR]

This article discusses various issues related to site-specific polymer attachment to a CCL-5 analogue by oxime exchange. The article authors have developed an improved synthetic strategy that allows effective protection of the aminooxy group during peptide synthesis, peptide ligation, and protein folding. Mild deprotection of the resulting proteins in solution occurs without interference with the native folded structure for subsequent specific attachment of a modifier such as a polymer. They applied this approach to the site-specific attachment of poly ethylene glycol (PEG) to a synthetic CCL-5 analogue. The objective was to attach a PEG polymer in the GAG binding site at position 45 to form CCL-5-PEG, to reduce aggregation and to increase the circulating lifetime.