Improving Stability Enhances In Vivo Efficacy of a PCSK9 Inhibitory Peptide.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

The emerging landscape of peptide-based inhibitors of PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating cardiovascular disease (CVD) due to its involvement in cholesterol metabolism. Although approved monoclonal antibodies (alirocumab and evolocumab) that inhibit PCSK9 function are very effective in lowering cholesterol, their limitations, including high treatment costs, have so far prohibited widespread use. Accordingly, there is great interest in alternative drug modalities to antibodies. Like antibodies, peptides are valuable therapeutics due to their high target potency and specificity. Furthermore, being smaller than antibodies means they have access to more drug administration options, are less likely to induce adverse immunogenic responses, and are better suited to affordable production. This review surveys the current peptide-based landscape aimed towards PCSK9 inhibition, covering pre-clinical to patented drug candidates and comparing them to current cholesterol lowering therapeutics. Classes of peptides reported to be inhibitors include nature-inspired disulfide-rich peptides, combinatorially derived cyclic peptides, and peptidomimetics. Their functional activities have been validated in biophysical and cellular assays, and in some cases pre-clinical mouse models. Recent efforts report peptides with potent sub-nanomolar binding affinities to PCSK9, which highlights their potential to achieve antibody-like potency. Studies are beginning to address pharmacokinetic properties of PCSK9-targeting peptides in more detail. We conclude by highlighting opportunities to investigate their biological effects in pre-clinical models of cardiovascular disease. The anticipation concerning the PCSK9-targeting peptide landscape is accelerating and it seems likely that a peptide-based therapeutic for treating PCSK9-mediated hypercholesterolemia may be clinically available in the near future.

Increased Valency Improves Inhibitory Activity of Peptides Targeting Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9).

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating hypercholesterolemia. Peptide-based PCSK9 inhibitors have attracted pharmaceutical interest, but the effect of multivalency on bioactivity is poorly understood. Here we designed bivalent and tetravalent dendrimers, decorated with the PCSK9 inhibitory peptides Pep2-8[RRG] or P9-38, to study relationships between peptide binding affinity, peptide valency, and PCSK9 inhibition. Increased valency resulted in improved PCSK9 inhibition for both peptides, with activity improvements of up to 100-fold achieved for the P9-38-decorated dendrimers compared to monomeric P9-38 in in vitro competition binding assays. Furthermore, the P9-38-decorated dendrimers showed improved potency at restoring functional low-density lipoprotein (LDL) receptor levels and internalizing LDL in the presence of PCSK9, demonstrating significant cell-based activity at picomolar concentrations. This study demonstrates the potential of increasing valency as a strategy for increasing the efficacy of peptide-based PCSK9 therapeutics.

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis.

Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and cystine knot framework, which engenders them with remarkably high stability. This study reports the cystine knot-related peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the cystine knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.

Engineered EGF-A Peptides with Improved Affinity for Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9).

The epidermal growth-factor-like domain A (EGF-A) of the low-density lipoprotein (LDL) receptor is a promising lead for therapeutic inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9). However, the clinical potential of EGF-A is limited by its suboptimal affinity for PCSK9. Here, we use phage display to identify EGF-A analogues with extended bioactive segments that have improved affinity for PCSK9. The most potent analogue, TEX-S2_03, demonstrated ∼130-fold improved affinity over the parent domain and had a reduced calcium dependency for efficient PCSK9 binding. Thermodynamic binding analysis suggests the improved affinity of TEX-S2_03 is enthalpically driven, indicating favorable interactions are formed between the extended segment of TEX-S2_03 and the PCSK9 surface. The improved affinity of TEX-S2_03 resulted in increased activity in competition binding assays and more efficient restoration of LDL receptor levels with clearance of extracellular LDL cholesterol in functional cell assays. These results confirm that TEX-S2_03 is a promising therapeutic lead for treating hypercholesterolemia. Many EGF-like domains are involved in disease-related protein-protein interactions; therefore, our strategy for engineering EGF-like domains has the potential to be broadly implemented in EGF-based drug design.

Bioactive Cyclization Optimizes the Affinity of a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Peptide Inhibitor.

Peptides are regarded as promising next-generation therapeutics. However, an analysis of over 1000 bioactive peptide candidates suggests that many have underdeveloped affinities and could benefit from cyclization using a bridging linker sequence. Until now, the primary focus has been on the use of inert peptide linkers. Here, we show that affinity can be significantly improved by enriching the linker with functional amino acids. We engineered a peptide inhibitor of PCSK9, a target for clinical management of hypercholesterolemia, to demonstrate this concept. Cyclization linker optimization from library screening produced a cyclic peptide with ∼100-fold improved activity over the parent peptide and efficiently restored low-density lipoprotein (LDL) receptor levels and cleared extracellular LDL. The linker forms favorable interactions with PCSK9 as evidenced by thermodynamics, structure-activity relationship (SAR), NMR, and molecular dynamics (MD) studies. This PCSK9 inhibitor is one of many peptides that could benefit from bioactive cyclization, a strategy that is amenable to broad application in pharmaceutical design.

EGF-like and Other Disulfide-rich Microdomains as Therapeutic Scaffolds.

Disulfide bonds typically introduce conformational constraints into peptides and proteins, conferring improved biopharmaceutical properties and greater therapeutic potential. In our opinion, disulfide-rich microdomains from proteins are potentially a rich and under-explored source of drug leads. A survey of the UniProt protein database shows that these domains are widely distributed throughout the plant and animal kingdoms, with the EGF-like domain being the most abundant of these domains. EGF-like domains exhibit large diversity in their disulfide bond topologies and calcium binding modes, which we classify in detail here. We found that many EGF-like domains are associated with disease phenotypes, and the interactions they mediate are potential therapeutic targets. Indeed, EGF-based therapeutic leads have been identified, and we further propose that these domains can be optimized to expand their therapeutic potential using chemical design strategies. This Review highlights the potential of disulfide-rich microdomains as future peptide therapeutics.