Integrin Subtypes and Nanoscale Ligand Presentation Influence Drug Sensitivity in Cancer Cells.

Cancer cell-matrix interactions have been shown to enhance cancer cell survival via the activation of pro-survival signaling pathways. These pathways are initiated at the site of interaction, i.e., integrins, and thus, their inhibition has been the target of therapeutic strategies. Individual roles for fibronectin-binding integrin subtypes αvß3 and α5ß1 have been shown for various cellular processes; however, a systematic comparison of their function in adhesion-dependent chemoresistance is lacking. Here, we utilize integrin subtype-specific peptidomimetics for αvß3 and α5ß1, both as blocking agents on fibronectin-coated surfaces and as surface-immobilized adhesion sites, in order to parse out their role in breast cancer cell survival. Block copolymer micelle nanolithography is utilized to immobilize peptidomimetics onto highly ordered gold nanoparticle arrays with biologically relevant interparticle spacings (35, 50, or 70 nm), thereby providing a platform for ascertaining the dependence of ligand spacing in chemoprotection. We show that several cellular properties-morphology, focal adhesion formation, and migration-are intricately linked to both the integrin subtype and their nanospacing. Importantly, we show that chemotherapeutic drug sensitivity is highly dependent on both parameters, with smaller ligand spacing generally hindering survival. Furthermore, we identify ligand type-specific patterns of drug sensitivity, with enhanced chemosurvival when cells engage αvß3 vs α5ß1 on fibronectin; however, this is heavily reliant on nanoscale spacing, as the opposite is observed when ligands are spaced at 70 nm. These data imply that even nanoscale alterations in extracellular matrix properties have profound effects on cancer cell survival and can thus inform future therapies and drug testing platforms.

Novel cilengitide-based cyclic RGD peptides as αvß3 integrin inhibitors.

In this letter, we report a series of five new RGD-containing cyclic peptides as potent inhibitors to αvß3 integrin protein. We have incorporated various unnatural lipophilic amino acids into the cyclic RGD framework of cilengitide, which is selective for αvß3 integrin. All the newly synthesized cyclic peptides were evaluated in vitrosolid phase binding assay and investigated for their bindingbehaviourtowards integrin subtypes. All the cyclic peptides were synthesized in excellent yield following solution-phase coupling strategy. The cyclic RGD peptides 1a-e exhibited IC50 of 9.9, 5.5, 72, 11 and 3.3 nM, respectively, towardsαvß3 integrin protein. This finding offers further opportunities for the introduction unusual amino acids into the cyclic peptide framework of cilengitide.

Bioconjugation of Supramolecular Metallacages to Integrin Ligands for Targeted Delivery of Cisplatin.

Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.

Enhancing Oral Bioavailability of Cyclic RGD Hexa-peptides by the Lipophilic Prodrug Charge Masking Approach: Redirection of Peptide Intestinal Permeability from a Paracellular to Transcellular Pathway.

Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-fold, indicating paracellular permeability. The Papp of the prodrug derivatives was much higher than that of their parent peptides. For instance, the Papp of the prodrug 12P was 20-fold higher than the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.

Improving oral bioavailability of cyclic peptides by N-methylation.

The renaissance of peptides in pharmaceutical industry results from their importance in many biological functions. However, low metabolic stability and the lack of oral availability of most peptides is a certain limitation. Whereas metabolic instability may be often overcome by development of small cyclic peptides containing d-amino acids, the very low oral availability of most peptides is a serious limitation for some medicinal applications. The situation is complicated because a twofold optimization - biological activity and oral availability - is required to overcome this problem. Moreover, most simple "rules" for achieving oral availability are not general and are applicable only to limited cases. Many structural modifications for increasing biological activities and metabolic stabilities of cyclic peptides have been described, of which N-alkylation is probably the most common. This mini-review focuses on the effects of N-methylation of cyclic peptides in strategies to optimize bioavailabilities.

Orally Active Peptides: Is There a Magic Bullet?

For decades, the development of peptides as potential drugs was aimed solely at peptides with the highest affinity, receptor selectivity, or stability against enzymatic degradation. However, optimization of their oral availability is highly desirable to establish orally active peptides as potential drug candidates for everyday use. A twofold optimization process is necessary to produce orally active peptides: 1) optimization of the affinity and selectivity and 2) optimization of the oral availability. These two steps must be performed sequentially for the rational design of orally active peptides. Nevertheless, additional knowledge is required to understand which structural changes increase oral availability, followed by incorporation of these elements into a peptide without changing its other biological properties. Considerable efforts have been made to understand the influence of these modifications on oral availability. One approach is to improve the oral availability of a peptide that has been previously optimized for biological activity, as described in (1) above. The second approach is to first identify an intestinally permeable, metabolically stable peptide scaffold and then introduce the functional groups necessary for the desired biological function. Previous approaches to achieving peptide oral availability have been claimed to have general applicability but, thus far, most of these solutions have not been successful in other cases. This Review discusses diverse chemical modifications, model peptides optimized for bioavailability, and orally active peptides to summarize the state of the research on the oral activity of peptides. We explain why no simple and straightforward strategy (i.e. a "magic bullet") exists for the design of an orally active peptide with a druglike biological function.

From a Helix to a Small Cycle: Metadynamics-Inspired αvß6 Integrin Selective Ligands.

The RGD-recognizing αvß6 integrin has only recently emerged as a major target for cancer diagnosis and therapy. Thus, the development of selective, low-molecular-weight ligands of this receptor is still in great demand. Here, a metadynamics-driven design strategy allowed us to successfully convert a helical nonapeptide into a cyclic pentapeptide (6) showing remarkable potency and αvß6 specificity. NMR and docking studies elucidated the reasons for the high affinity and selectivity of this compound, setting the ground for the rational design of new αvß6-specific small peptides or even peptidomimetics. In vivo PET imaging studies demonstrated the potential use of 6 for medical applications.

Overcoming the Lack of Oral Availability of Cyclic Hexapeptides: Design of a Selective and Orally Available Ligand for the Integrin†αvß3.

A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrin αvß3 is based on two concepts: a) screening of systematically designed libraries with spatial diversity and b) masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1) initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2) selection of cyclic peptides with the highest intestinal permeability; 3) design of sublibraries with the bioactive RGD sequence in all possible positions; 4) selection of the best ligands for RGD-recognizing integrin subtypes; 5) fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6) protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7) proof of biological effects in mice after oral administration.

Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery.

In this work we report synthesis and biological evaluation of a cell-penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne-azide click reaction. Cell viability studies in several cell-lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF-7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The organometallic ferrocene exhibits amplified anti-tumor activity by targeted delivery via highly selective ligands to αvß3, αvß6, or α5ß1 integrins.

High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvß3, α5ß1, or αvß6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvß3 (L2b) and an αvß6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.

Surface Co-presentation of BMP-2 and integrin selective ligands at the nanoscale favors α5ß1 integrin-mediated adhesion.

Here we present the use of surface nanopatterning of covalently immobilized BMP-2 and integrin selective ligands to determine the specificity of their interactions in regulating cell adhesion and focal adhesion assembly. Gold nanoparticle arrays carrying single BMP-2 dimers are prepared by block-copolymer micellar nanolithography and azide-functionalized integrin ligands (cyclic-RGD peptides or α5ß1 integrin peptidomimetics) are immobilized on the surrounding polyethylene glycol alkyne by click chemistry. Compared to BMP-2 added to the media, surface immobilized BMP-2 (iBMP-2) favors the spatial segregation of adhesion clusters and enhances focal adhesion (FA) size in cells adhering to α5ß1 integrin selective ligands. Moreover, iBMP-2 copresented with α5ß1 integrin ligands induces the recruitment of αvß3 integrins in FAs. When copresented with RGD, iBMP-2 induces the assembly of a higher number of FAs, which are not affected by α5ß1 integrin blocking. Our dual-functionalized platforms offer the possibility to study the crosstalk between integrins and BMP receptors, and more in general they could be used to address the spatial regulation of growth factors and adhesion receptors crosstalk on biomimetic surfaces.

Halting the Spread of Herpes Simplex Virus-1: The Discovery of an Effective Dual αvß6/αvß8 Integrin Ligand.

Over recent years, αvß6 and αvß8 Arg-Gly-Asp (RGD) integrins have risen to prominence as interchangeable co-receptors for the cellular entry of herpes simplex virus-1 (HSV-1). In fact, the employment of subtype-specific integrin-neutralizing antibodies or gene-silencing siRNAs has emerged as a valuable strategy for impairing HSV infectivity. Here, we shift the focus to a more affordable pharmaceutical approach based on small RGD-containing cyclic pentapeptides. Starting from our recently developed αvß6-preferential peptide [RGD-Chg-E]-CONH2 (1), a small library of N-methylated derivatives (2-6) was indeed synthesized in the attempt to increase its affinity toward αvß8. Among the novel compounds, [RGD-Chg-(NMe)E]-CONH2 (6) turned out to be a potent αvß6/αvß8 binder and a promising inhibitor of HSV entry through an integrin-dependent mechanism. Furthermore, the renewed selectivity profile of 6 was fully rationalized by a NMR/molecular modeling combined approach, providing novel valuable hints for the design of RGD integrin ligands with the desired specificity profile.

Organoids to Study Intestinal Nutrient Transport, Drug Uptake and Metabolism - Update to the Human Model and Expansion of Applications.

Intestinal transport and sensing processes and their interconnection to metabolism are relevant to pathologies such as malabsorption syndromes, inflammatory diseases, obesity and type 2 diabetes. Constituting a highly selective barrier, intestinal epithelial cells absorb, metabolize, and release nutrients into the circulation, hence serving as gatekeeper of nutrient availability and metabolic health for the whole organism. Next to nutrient transport and sensing functions, intestinal transporters including peptide transporter 1 (PEPT1) are involved in the absorption of drugs and prodrugs, including certain inhibitors of angiotensin-converting enzyme, protease inhibitors, antivirals, and peptidomimetics like ß-lactam antibiotics. Here, we verify the applicability of 3D organoids for in vitro investigation of intestinal biochemical processes related to transport and metabolism of nutrients and drugs. Establishing a variety of methodologies including illustration of transporter-mediated nutrient and drug uptake and metabolomics approaches, we highlight intestinal organoids as robust and reliable tool in this field of research. Currently used in vitro models to study intestinal nutrient absorption, drug transport and enterocyte metabolism, such as Caco-2 cells or rodent explant models are of limited value due to their cancer and non-human origin, respectively. Particularly species differences result in poorly correlative data and findings obtained in these models cannot be extrapolated reliably to humans, as indicated by high failure rates in drug development pipelines. In contrast, human intestinal organoids represent a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement and Replacement) principle in basic science as well as the preclinical and regulatory setup.

Selective Targeting of Integrin αvß8 by a Highly Active Cyclic Peptide.

Integrins play important roles in physiological and pathophysiological processes. Among the RGD-recognizing integrin subtypes, the αvß8 receptor is emerging as an attractive target because of its involvement in various illnesses, such as autoimmune diseases, viral infections, and cancer. However, its functions have, so far, not been investigated in living subjects mainly because of the lack of a selective αvß8 ligand. Here, we report the design and potential medical applications of a cyclic octapeptide as the first highly selective small-molecule ligand for αvß8. Remarkably, this compound displays low nanomolar αvß8 binding affinity and a strong discriminating power of at least 2 orders of magnitude versus other RGD-recognizing integrins. Peptide functionalization with fluorescent or radioactive labels enables the selective imaging of αvß8-positive cells and tissues. This new probe will pave the way for detailed characterization of the distinct (patho)physiological role of this relatively unexplored integrin, providing a basis to fully exploit the potential of αvß8 as a target for molecular diagnostics and personalized therapy regimens.

Therapeutic Radiopharmaceuticals Targeting Integrin αvß6.

The epithelial integrin αvß6 is expressed by many malignant carcinoma cell types, including pancreatic cancer, and thus represents a promising target for radionuclide therapy. The peptide cyclo(FRGDLAFp(NMe)K) was decorated with different chelators (DOTPI, DOTAGA, and DOTA). The Lu(III) complexes of these conjugates exhibited comparable αvß6 integrin affinities (IC50 ranging from 0.3 to 0.8 nM) and good selectivities against other integrins (IC50 for αvß8 >43 nM; for α5ß1 >238 nM; and for αvß3, αvß5, and αIIbß3 >1000 nM). Although different formal charges of the Lu(III) chelates (ranging from 0 to 4) resulted in strongly varying degrees of hydrophilicity (log D ranging from -3.0 to -4.1), biodistributions in murine H2009 xenografts of the Lu-177-labeled compounds (except the DOTPI derivative) were quite similar and comparable to our previously reported αvß6 integrin positron emission tomography tracer Ga-68-avebehexin. Hence, combinations of existing Ga-68- and Lu-177-labeled c(FRGDLAFp(NMe)K) derivatives could be utilized for αvß6 integrin-targeted theranostics, whereas our data nonetheless suggest that further improvement of pharmacokinetics might be necessary to ensure clinical success.

N-Methylation of isoDGR Peptides: Discovery of a Selective α5ß1-Integrin Ligand as a Potent Tumor Imaging Agent.

Specific targeting of the integrin subtype α5ß1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective α5ß1/αvß6 peptide c(phg- isoDGR-k) into a potent peptidic RGD binding α5ß1 subtype selective ligand c(phg- isoDGR-( NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg- isoDGR-( NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring α5ß1 integrin expression in a M21 mouse xenograft.

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5ß1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5ß1/αvß3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5ß1/αvß3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5ß1 integrin.

Exploring the Role of RGD-Recognizing Integrins in Cancer.

Integrins are key regulators of communication between cells and with their microenvironment. Eight members of the integrin superfamily recognize the tripeptide motif Arg-Gly-Asp (RGD) within extracelluar matrix (ECM) proteins. These integrins constitute an important subfamily and play a major role in cancer progression and metastasis via their tumor biological functions. Such transmembrane adhesion and signaling receptors are thus recognized as promising and well accessible targets for novel diagnostic and therapeutic applications for directly attacking cancer cells and their fatal microenvironment. Recently, specific small peptidic and peptidomimetic ligands as well as antibodies binding to distinct integrin subtypes have been developed and synthesized as new drug candidates for cancer treatment. Understanding the distinct functions and interplay of integrin subtypes is a prerequisite for selective intervention in integrin-mediated diseases. Integrin subtype-specific ligands labelled with radioisotopes or fluorescent molecules allows the characterization of the integrin patterns in vivo and later the medical intervention via subtype specific drugs. The coating of nanoparticles, larger proteins, or encapsulating agents by integrin ligands are being explored to guide cytotoxic reagents directly to the cancer cell surface. These ligands are currently under investigation in clinical studies for their efficacy in interference with tumor cell adhesion, migration/invasion, proliferation, signaling, and survival, opening new treatment approaches in personalized medicine.