Peptide Scaffolds Research Articles

Osteopontine-derived functional fragments coupled to RADA16 self-assembled peptide hydrogels promotes bone and vascular regeneration in vivo

Biomaterial scaffolds have been widely used in tissue engineering. A functionalized self-assembled peptide scaffold named RADA16-OPD was designed by linking the short functional motif of osteopontine (OPN)-derived functional fragments SVVYGLR (OPD) to the C-terminus of the self-assembled peptide RADA16. Atomic force microscopy (AFM) was used to analyze the self-assembling peptide’s structural composition. The live/dead staining results showed that RADA16-OPD is not toxic to rASC. After creating a rat skull defect model artificially, micro-CT results revealed that the defect area treated with RADA16-OPD hydrogel had higher bone volume/total volume (BV/TV), a higher trabecular number (TB.N.), and higher bone density (BMD) at different treatment time points. Histological evaluation found that there was more new bone and mature collagen production in the RADA16-OPD group. Meanwhile, the RADA16-OPD group had higher expression of alkaline phosphatase (ALP) and osteocalcin (OCN) than the other two groups. Additionally, immunofluorescence revealed that the RADA16-OPD group had higher levels of platelet/endothelial cell adhesion molecule 1 (CD31) expression than the other two groups. It demonstrated the potential for clinical use of the RADA16-OPD peptide scaffold by promoting bone regeneration and blood vessel development in vivo.

Development of Antibacterial Peptides with Membrane Disruption and Folate Pathway Inhibitory Activities against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) offer an opportunity to overcome multidrug resistance. Here, novel peptides were designed based on AMP fragments derived from sea cucumber hemolytic lectin to enhance anti-methicillin-resistant Staphylococcus aureus (MRSA) activity with less side effects. Two designed peptides, CGS19 (LARVARRVIRFIRRAW-NH2) and CGS20 (RRRLARRLIFFIRRAW-NH2), exhibited strong antibacterial activities against clinically isolated MRSA with MICs of 3-6 μM, but no obvious cytotoxicity was observed. Consistently, CGS19 and CGS20 exerted rapid bactericidal activity and effectively induced 5.9 and 5.8 log reduction of MRSA counts in mouse subeschar, respectively. Further, CGS19 and CGS20 kill bacteria not only through disturbing membrane integrity but also by binding formate-tetrahydrofolate ligase, a key enzyme in the folate metabolism pathway, thereby inhibiting the folate pathway of MRSA. CGS19 and CGS20 are promising lead candidates for drug development against MRSA infection. The dual mechanisms on the identical peptide sequence or scaffold might be an underappreciated manner of treating life-threatening pathogens.

Precision peptide theranostics: developing N- to C-terminus optimized theranostics targeting cholecystokinin-2 receptor.

Peptides are ideal for theranostic development as they afford rapid target accumulation, fast clearance from background tissue, and exhibit good tissue penetration. Previously, we developed a novel series of peptides that presented discreet folding propensity leading to an optimal candidate [68Ga]Ga-DOTA-GA1 ([D-Glu]6-Ala-Tyr-NMeGly-Trp-NMeNle-Asp-Nal-NH2) with 50 pM binding affinity against cholecystokinin-2 receptors (CCK2R). However, we were confronted with challenges of unfavorably high renal uptake. Methods: A structure activity relationship study was undertaken of the lead theranostic candidate. Prudent structural modifications were made to the peptide scaffold to evaluate the contributions of specific N-terminal residues to the overall biological activity. Optimal candidates were then evaluated in nude mice bearing transfected A431-CCK2 tumors, and their biodistribution was quantitated ex vivo. Results: We identified and confirmed that D-Glu3 to D-Ala3 substitution produced 2 optimal candidates, [68Ga]Ga-DOTA-GA12 and [68Ga]Ga-DOTA-GA13. These radiopeptides presented with high target/background ratios, enhanced tumor retention, excellent metabolic stability in plasma and mice organ homogenates, and a 4-fold reduction in renal uptake, significantly outperforming their non-alanine counterparts. Conclusions: Our study identified novel radiopharmaceutical candidates that target the CCK2R. Their high tumor uptake and reduced renal accumulation warrant clinical translation.

Modulation of Ferrocene–Ferrocene Interactions by Varying Their Reciprocal Positions in L-Dap/Aib Helical Peptides

In this work, we developed two new polyfunctional hybrid systems in which the presence of Fc redox “antennas” on peptide scaffolds allows for a modulation of their electronic properties. Specifically, we synthesized two helical hexapeptides containing four Aib (α-amionoisobutyric acid) and two L-Dap (2,3-diamino propionic acid) residues. L-Dap side chains were then functionalized with Fc moieties. The structures of the two 310 helical peptides, namely Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr and Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, were investigated by X-ray diffraction, 2D-NMR, CD and IR spectroscopies. Due to the helical conformation, in Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr, the Fc groups are located on the same face of the helix, but in Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, they are located on opposite faces. Surprisingly, two bands were found through DPV for Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, indicating an electrostatic interaction between the Fc groups despite their longer reciprocal distance with respect to that in Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr. CD experiments at different concentrations evidenced aggregation for Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, even at high dilutions, thus suggesting that the Fc-Fc electrostatic interaction could be of an intermolecular nature.

Bionic peptide scaffold in situ polarization and recruitment of M2 macrophages to promote peripheral nerve regeneration.

Tissue regeneration requires exogenous and endogenous signals, and there is increasing evidence that the exogenous microenvironment may play an even more dominant role in the complex process of coordinated multiple cells. The short-distance peripheral nerve showed a spontaneous regenerative phenomenon, which was initiated by the guiding role of macrophages. However, it cannot sufficiently restore long-distance nerve injury by itself. Based on this principle, we firstly constructed a proinflammatory model to prove that abnormal M2 expression reduce the guidance and repair effect of long-distance nerves. Furthermore, a bionic peptide hydrogel scaffold based on self-assembly was developed to envelop M2-derived regenerative cytokines and extracellular vesicles (EVs). The cytokines and EVs were quantified to mimic the guidance and regenerative microenvironment in a direct and mild manner. The bionic scaffold promoted M2 transformation in situ and led to proliferation and migration of Schwann cells, neuron growth and motor function recovery. Meanwhile, the peptide scaffold combined with CX3CL1 recruited more blood-derived M2 macrophages to promote long-distance nerve reconstruction. Overall, we systematically confirmed the important role of M2 in regulating and restoring the injury peripheral nerve. This bionic peptide hydrogel scaffold mimicked and remodeled the local environment for M2 transformation and recruitment, favoring long-distance peripheral nerve regeneration. It can help to explicate regulative effect of M2 may be a cause not just a consequence in nerve repair and tissue integration, which facilitating the development of pro-regenerative biomaterials.

Calming the Nerves via the Immune Instructive Physiochemical Properties of Self-Assembling Peptide Hydrogels.

Current therapies for the devastating damage caused by traumatic brain injuries (TBI) are limited. This is in part due to poor drug efficacy to modulate neuroinflammation, angiogenesis and/or promoting neuroprotection and is the combined result of challenges in getting drugs across the blood brain barrier, in a targeted approach. The negative impact of the injured extracellular matrix (ECM) has been identified as a factor in restricting post-injury plasticity of residual neurons and is shown to reduce the functional integration of grafted cells. Therefore, new strategies are needed to manipulate the extracellular environment at the subacute phase to enhance brain regeneration. In this review, potential strategies are to be discussed for the treatment of TBI by using self-assembling peptide (SAP) hydrogels, fabricated via the rational design of supramolecular peptide scaffolds, as an artificial ECM which under the appropriate conditions yields a supramolecular hydrogel. Sequence selection of the peptides allows the tuning of these hydrogels' physical and biochemical properties such as charge, hydrophobicity, cell adhesiveness, stiffness, factor presentation, degradation profile and responsiveness to (external) stimuli. This review aims to facilitate the development of more intelligent biomaterials in the future to satisfy the parameters, requirements, and opportunities for the effective treatment of TBI.

Designing Cardin-Motif Peptide and Heparin-Based Multicomponent Advanced Bioactive Hydrogel Scaffolds to Control Cellular Behavior.

Recently, peptide and sugar-based multicomponent systems have gained much interest in attaining the sophisticated structure and biofunctional complexity of the extracellular matrix (ECM). To this direction, we have designed for the first time a biologically relevant minimalist Cardin-motif peptide capable of binding ECM-derived glycosaminoglycans. Herein, we explored Cardin-motif peptide and heparin-based biomolecular matrix by employing simple noncovalent interactions at the molecular level. Interestingly, this peptide was inadequate to induce hydrogelation at ambient pH due to the presence of basic amino acids. However, addition of heparin successfully triggered its gelation at physiological pH following favorable electrostatic interactions with heparin. Importantly, the newly developed scaffolds displayed tunable nanofibrous morphology and superior mechanical properties as controlled simply by the differential mixing ratio of both biomolecular entities. Additionally, these composite scaffolds could closely mimic the complexity of ECM as they demonstrated superior biocompatibility and enhanced growth and proliferation of neural cells as compared to the peptide scaffold.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 μM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

Differences in heavy metal binding to cysteine-containing coiled-coil peptides.

One third of all structurally characterised proteins contain a metal; however, the interplay between metal-binding and peptide/protein folding has yet to be fully elucidated. To better understand how metal binding affects peptide folding, a range of metals should be studied within a specific scaffold. To this end, we modified a histidine-containing coiled-coil peptide to create a cysteine-containing scaffold, named CX3C, which was designed to bind heavy metal ions. In addition, we generated a peptide named CX2C, which contains a binding site more commonly found in natural proteins. Using a combination of analytical techniques including circular dichroism (CD) spectroscopy, UV-Vis spectroscopy and size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we examined the differences in the metal-binding properties of the two peptides. Both peptides are largely unfolded in the apo state due to the disruption of the hydrophobic core by inclusion of the polar cysteine residues. However, this unfolding is overcome by the addition of Cd(II), Pb(II) and Hg(II), and helical assemblies are formed. Both peptides have differing affinities for these metal ions, a fact likely attributed to the differing sizes of the ions. We also show that the oligomerisation state of the peptide complexes and the coordination geometries of the metal ions differ between the two peptide scaffolds. These findings highlight that subtle changes in the primary structure of a peptide can have considerable implications for metal binding.

Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited β-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity drugs.

Advances in MRI: Peptide and peptidomimetic-based contrast agents.

Magnetic resonance imaging (MRI) is a common medical imaging technique that provides three-dimensional body images. MRI contrast agents improve image contrast by raising the rate of water proton relaxation in specific tissues. Peptides and peptidomimetics act as scaffolds for MRI imaging agents because of their increased size and offer the possibility to engine a higher hydration value within the design. The design of a new Gd-based contrast agent must take into account high stability constants to avoid free Gd(III), with the subsequent nephrotoxicity, and high relaxivity values. This review analyzes various synthetic approaches, reports studies of relaxometric parameters, and focuses on the description and application of Gd(III)-chelates based on peptide and peptidomimetic scaffolds. In addition, the X-ray molecular structures of three DOTA complexes will be reported to emphasize the necessity of using the X-ray diffraction analysis to identify the coordination sphere of the metals and the mechanism of action of the compounds.

Biomimetic Electrospun Self-Assembling Peptide Scaffolds for Neural Stem Cell Transplantation in Neural Tissue Engineering.

Spinal cord regeneration using stem cell transplantation is a promising strategy for regenerative therapy. Stem cells transplanted onto scaffolds that can mimic natural extracellular matrix (ECM) have the potential to significantly improve outcomes. In this study, we strived to develop a cell carrier by culturing neural stem cells (NSCs) onto electrospun 2D and 3D constructs made up of specific crosslinked functionalized self-assembling peptides (SAPs) featuring enhanced biomimetic and biomechanical properties. Morphology, architecture, and secondary structures of electrospun scaffolds in the solid-state and electrospinning solution were studied step by step. Morphological studies showed the benefit of mixed peptides and surfactants as additives to form thinner, uniform, and defect-free fibers. It has been observed that β-sheet conformation as evidence of self-assembling has been predominant throughout the process except for the electrospinning solution. In vitro NSCs seeded on electrospun SAP scaffolds in 2D and 3D conditions displayed desirable proliferation, viability, and differentiation in comparison to the gold standard. In vivo biocompatibility assay confirmed the permissibility of implanted fibrous channels by foreign body reaction. The results of this study demonstrated that fibrous 2D/3D electrospun SAP scaffolds, when shaped as micro-channels, can be suitable to support NSC transplantation for regeneration following spinal cord injury.

Spatial Distribution and Biochemical Characterization of Serine Peptidase Inhibitors in the Venom of the Brazilian Sea Anemone Anthopleura cascaia Using Mass Spectrometry Imaging.

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Synthesis, characterization and evaluation of anti-hyperalgesia, anticonvulsant and antioxidant activity of novel VV-hemorphin-5 analogs.

Two series of new VV-hemorphin-5 analogs with structures Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH2 and Adam-Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH2 , where Xxx is Ac5c (1-aminocyclopentane-1-carboxylic acid), Ac6c (1-aminocyclohexane-1-carboxylic acid), Ac7c (1-aminocycloheptane-1-carboxylic acid), and Adam is the low-molecular-weight lipophilic adamantyl building block, were synthesized, characterized electrochemically and evaluated for antioxidant, anti-hyperalgesia, and anticonvulsant activity. The design of the compounds followed the strategy to improve the propensity for aqueous solubility and/or to increase their affinity for the target receptor or enzyme. The partition coefficient value shows that the peptide scaffold goes from hydrophilic to lipophilic with the increasing size of the cycloalkane ring and even more with the introduction of the adamantane. The peptides C5-V and C7-V were the only analogs that provoked an immediate antinociceptive effect changing the mechanical pain threshold. The six new peptide analogs produced a significant and long-lasting carrageenan model of inflammatory pain in rats. While the adamantane hemorphin analog Ad7-V was the only compound with the potency to suppress psychomotor seizures in the 6-Hz test, the C6-V and Ad6-V exhibited protective activity against the seizure spread in the maximal electroshock seizure test in mice. The active analogs did not show neurotoxicity or sedative effects. Our results revealed a structure-related specific activity of a newly designed hemorphin analog that could be used as a template for future modification and preparation of compounds with potential analgesic and anticonvulsant activity.

Concise Total Synthesis and Biological Evaluation of Pargamicin A and its Diastereomer, Piperazic Acid-containing Cyclopeptides.

We have accomplished the total synthesis, structure determination, and biological evaluation of pargamicin A and one of its diastereomers. Two key tripeptide segments were synthesized using a linear peptide elongation process that includes the direct coupling of a poorly nucleophilic piperazic acid derivative. The resulting tripeptides were coupled using triphosgene/collidine at ambient temperature leading to a precursor for the final cyclization step. T3P-promoted macrolactamization under high-dilution conditions, followed by the removal of the benzyl protecting group was used to furnish two putative structures of pargamicin A. Comparison of the 1 H and 13 C NMR spectra and the antibacterial activity of the natural and synthetic products successfully revealed that the absolute configuration of the N-hydroxy-Ile residue of pargamicin A is 2S,3S. A biological evaluation of synthetically obtained pargamicin A and its diastereomer suggested that the stereostructure of the cyclic peptide scaffold of the natural product plays a crucial role in determining the strength of its antibacterial activity.

Chemoselective Late-Stage Functionalization of Peptides via Photocatalytic C2-Alkylation of Tryptophan.

Across eukaryotic proteomes, tryptophan is the least abundant of the 20 canonical amino acids, which makes it an ideal chemical handle for the late-stage functionalization of peptide and protein scaffolds with minimal production of undesired isoforms. Herein, we report the photocatalytic C2-alkylation of tryptophan using bromodifluoroacetate/acetamide-derived radical precursors. This rapid visible-light-mediated reaction is additive-free, operationally simple, and tolerates diverse functionality. We demonstrate the late-stage modification of a variety of complex peptides, including examples of biological significance.

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

Recent Advances in Formulations for Long-Acting Delivery of Therapeutic Peptides.

Recent preclinical and clinical studies have focused on the active area of therapeutic peptides due to their high potency, selectivity, and specificity in treating a broad range of diseases. However, therapeutic peptides suffer from multiple disadvantages, such as limited oral bioavailability, short half-life, rapid clearance from the body, and susceptibility to physiological conditions (e.g., acidic pH and enzymolysis). Therefore, high peptide dosages and dose frequencies are required for effective patient treatment. Recent innovations in pharmaceutical formulations have substantially improved therapeutic peptide administration by providing the following advantages: long-acting delivery, precise dose administration, retention of biological activity, and improvement of patient compliance. This review discusses therapeutic peptides and challenges in their delivery and explores recent peptide delivery formulations, including micro/nanoparticles (based on lipids, polymers, porous silicon, silica, and stimuli-responsive materials), (stimuli-responsive) hydrogels, particle/hydrogel composites, and (natural or synthetic) scaffolds. This review further covers the applications of these formulations for prolonged delivery and sustained release of therapeutic peptides and their impact on peptide bioactivity, loading efficiency, and (in vitro/in vivo) release parameters.

Unveiling the Role of Capping Groups in Naphthalene N-Capped Dehydrodipeptide Hydrogels.

Self-assembled peptide-based hydrogels are archetypical nanostructured materials with a plethora of foreseeable applications in nanomedicine and as biomaterials. N-protected di- and tri-peptides are effective minimalist (molecular) hydrogelators. Independent variation of the capping group, peptide sequence and side chain modifications allows a wide chemical space to be explored and hydrogel properties to be tuned. In this work, we report the synthesis of a focused library of dehydrodipeptides N-protected with 1-naphthoyl and 2-naphthylacetyl groups. The 2-naphthylacetyl group was extensively reported for preparation of peptide-based self-assembled hydrogels, whereas the 1-naphthaloyl group was largely overlooked, owing presumably to the lack of a methylene linker between the naphthalene aromatic ring and the peptide backbone. Interestingly, dehydrodipeptides N-capped with the 1-naphthyl moiety afford stronger gels, at lower concentrations, than the 2-naphthylacetyl-capped dehydrodipeptides. Fluorescence and circular dichroism spectroscopy showed that the self-assembly of the dehydrodipeptides is driven by intermolecular aromatic π-π stacking interactions. Molecular dynamics simulations revealed that the 1-naphthoyl group allows higher order aromatic π-π stacking of the peptide molecules than the 2-naphthylacetyl group, together with hydrogen bonding of the peptide scaffold. The nanostructure of the gel networks was studied by TEM and STEM microscopy and was found to correlate well with the elasticity of the gels. This study contributes to understanding the interplay between peptide and capping group structure on the formation of self-assembled low-molecular-weight peptide hydrogels. Moreover, the results presented here add the 1-naphthoyl group to the palette of capping groups available for the preparation of efficacious low-molecular-weight peptide-based hydrogels.

Differential Blood-Brain Barrier Transport and Cell Uptake of Cyclic Peptides In Vivo and In Vitro.

The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, Kp,uu,brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.