Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Double helix formation in α‐peptides: a theoretical study

A complete overview on all possible hydrogen bonding patterns of double helices with antiparallel and parallel strand orientation in α‐peptide sequences is provided on the basis of ab initio molecular orbital theory. The most stable representatives belong to the group of antiparallel helices. The study on side chain influence shows that these double helices can only be realized if the strands are composed of L ‐ and D ‐amino acids in alternate order. The stability of the double helices is compared with that of competing single‐stranded helices. The data contribute to an understanding of secondary structure formation in peptides and provide a basis for a rational design of membrane channels. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

β‐Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β‐aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β³‐ and/or β²‐amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β‐peptide substrates, β‐aminopeptidases have potential to be used as biocatalysts for β‐peptide synthesis and for the resolution of enantiomerically pure β‐amino acids from racemic substrate mixtures. β‐Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N‐terminus of the newly formed β‐polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N‐terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β‐aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β‐aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β‐peptide conversion.     

Synthesis and characterization of cyclic peptides that are β‐helical in trifluoroethanol

We show that three designed cyclic d ,l ‐peptides are β‐helical in TFE—a solvent in which the archetypal β‐helical peptide, gA, is unstructured. This result represents an advance in the field of β‐helical peptide foldamers and a step toward achieving β‐helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG–PS composite resin prefunctionalized with the alkanesulfonamide ‘SCL’ linker and preloaded with glycine. Our new conditions avoided C‐terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic β‐helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet–Spengler reaction of β3‐homo‐tryptophan derivatives with chiral α‐amino aldehydes

The Pictet–Spengler (PS) cyclizations of β³‐hTrp derivatives as arylethylamine substrates were performed with L‐α‐amino and D‐α‐amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3‐disubstituted 1,2,3,4‐tetrahydro‐β‐carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the β‐turn induction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and Biological Evaluation of Gramicidin S‐Inspired Cyclic Mixed α/β‐Peptides

Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evans' chiral acylated oxazolidinones the β²‐amino acids (R)‐ and (S)‐Fmoc‐β²homovaline and (R)‐Fmoc‐β²homoleucine are synthesized. These building blocks were used, in combination with commercially available α‐ and β³‐amino acids, for the synthesis of the cyclo‐(αβ³αβ²α)₂ peptide 2 and the cyclo‐(αβ²αβ³α)₂ peptides 3 – 5 . The peptides 2 – 5 were screened for their ability to inhibit a small panel of Gram‐negative and Gram‐positive bacterial strains.     

Analysis of protein transmembrane helical regions by a neural network.

Neural networks were used to generalize common themes found in transmembrane-spanning protein helices. Various-sized databases were used containing nonoverlapping sequences, each 25 amino acids long. Training consisted of sorting these sequences into 1 of 2 groups: transmembrane helical peptides or nontransmembrane peptides. Learning was measured using a test set 10% the size of the training set. As training set size increased from 214 sequences to 1,751 sequences, learning increased in a nonlinear manner from 75% to a high of 98%, then declined to a low of 87%. The final training database consisted of roughly equal numbers of transmembrane (928) and nontransmembrane (1,018) sequences. All transmembrane sequences were entered into the database with respect to their lipid membrane orientation: from inside the membrane to outside. Generalized transmembrane helix and nontransmembrane peptides were constructed from the maximally weighted connecting strengths of fully trained networks. Four generalized transmembrane helices were found to contain 9 consensus residues: a K-R-F triplet was found at the inside lipid interface, 2 isoleucine and 2 other phenylalanine residues were present in the helical body, and 2 tryptophan residues were found near the outside lipid interface. As a test of the training method, bacteriorhodopsin was examined to determine the position of its 7 transmembrane helices.     

Interaction of β3/β2‐Peptides,Consisting of Val‐Ala‐Leu Segments,with POPC Giant Unilamellar Vesicles (GUVs) and White Blood Cancer Cells (U937) – A New Type of Cell‐Penetrating Peptides,and a Surprising Chain‐Length Dependence of Their Vesicle‐ and Cell‐Lysing Activity

Many years ago, β²/β³‐peptides, consisting of alternatively arranged β²‐ and β³h‐amino‐acid residues, have been found to undergo folding to a unique type of helix, the 10/12‐helix, and to exhibit non‐polar, lipophilic properties (Helv. Chim. Acta 1997 , 80, 2033). We have now synthesized such ‘mixed’ hexa‐, nona‐, dodeca‐, and octadecapeptides, consisting of Val‐Ala‐Leu triads, with N‐terminal fluorescein (FAM) labels, i.e., 1 – 4 , and studied their interactions with POPC (=1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow‐cytometry assay, a membrane‐toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β³/β²‐peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β³/β²‐, β³‐, and β²‐nonamers, 2, 5 , and 6 , respectively, the derivatives 5 and 6 consisting exclusively of β³‐ or β²‐amino‐acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β³/β²‐nonamer and/or the β³/β²‐dodecamer derivative, 2 and/or 3 , respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host‐defense antimicrobial peptides. Possible sources of the chain‐length‐dependent destructive potential of the β³/β²‐nona‐ and β³/β²‐dodecapeptide derivatives, and a possible relationship with the phosphate‐to‐phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of ‘mixed’ β³/β²‐peptides with membranes and to evaluate possible biomedical applications.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational preferences of helix foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The conformational preferences of helix foldamers having different sizes of the H‐bonded pseudocycles have been studied for di‐ to octa‐γ^2,3‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α–C^β bond using density functional methods. The helical structures of the γAmc₆ oligopeptides with homochiral configurations are known to be much stable than those with heterochiral configurations in the gas phase and in solution (chloroform and water). In particular, it is found that the (P/M)?2.5₁₄‐helices are most preferred in the gas phase and in chloroform, whereas the (P/M)?2.3₁₂‐helices become most populated in water due to the larger helix dipole moments. As the peptide sequence becomes longer, the helix propensities of 14‐ and 12‐helices are found to increase both in the gas phase and in solution. The γAmc₆ peptides longer than octapeptide are expected to exist as a mixture of 12‐ and 14‐helices with the similar populations in water. The mean backbone torsion angles and helical parameters of the 14‐helix foldamers of γAmc₆ oligopeptides are quite similar to those of 2‐aminocyclohexylacetic acid oligopeptides and γ^2,3,4‐aminobutyric acid tetrapeptide in the solid state, despite the different substituents on the backbone. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 87–95, 2014.     

Exploring peptide-membrane interactions with coarse-grained MD simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

Folding thermodynamics of β‐hairpins studied by replica‐exchange molecular dynamics simulations

We study the differences in folding stability of β‐hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3‐1, and TrpZip4. By performing replica‐exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3‐1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β‐hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild‐type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state‐of‐the‐art all‐atom protein force fields. Proteins 2015; 83:1307–1315. © 2015 Wiley Periodicals, Inc.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

The β‐sheet breakers and π‐stacking

Fibrillation of β‐amyloid is recognized as a key process leading to the development of Alzheimer's disease. Small peptides called β‐sheet breakers were found to inhibit the process of β‐amyloid fibrillation and to dissolve amyloid fibrils in vitro, in vivo, and in cell culture studies [1,2]. The mechanism by which peptide inhibition takes place remains elusive and a detailed model needs to be established. Here, we present new insights into the possible role of consecutive Phe residues, present in the structure of β‐sheet breakers, supported by the results obtained by means of MD simulations. We performed a 30‐ns MD of two β‐sheet breakers: iAβ5 (LPFFD) and iAβ6 (LPFFFD) which have two and three consecutive Phe residues, respectively. We have found that Phe rings in these peptides tend to form stacked conformations. For one of the peptides – iAβ6 – the calculated electrostatic contribution to free energy of one of the conformers with three rings stacked (c2) is significantly lower than that corresponding to the unstacked one (c1), two rings stacked (c0) and second conformer with three rings stacked (c3). This may favor the interaction of the c2 conformer with the target on amyloid fibril. We hypothesize that the mechanism of inhibition of amyloidogenesis by β‐sheet breaker involves competition among π‐stacked Phe residues of the inhibitor and π‐stacking within the β‐amyloid fibril. iAβ6 may be a promising candidate for a lead compound of amyloidogenesis inhibitors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The determinants of hydrophobic mismatch response for transmembrane helices

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~ 45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.     

Analysis of ER stress in developing rice endosperm accumulating β‐amyloid peptide

The common neurodegenerative disorder known as Alzheimer’s disease is characterized by cerebral neuritic plaques of amyloid β (Aβ) peptide. Plaque formation is related to the highly aggregative property of this peptide, because it polymerizes to form insoluble plaques or fibrils causing neurotoxicity. Here, we expressed Aβ peptide as a new causing agent to endoplasmic reticulum (ER) stress to study ER stress occurred in plant. When the dimer of Aβ_1–42 peptide was expressed in maturing seed under the control of the 2.3‐kb glutelin GluB‐1 promoter containing its signal peptide, a maximum of about 8 μg peptide per grain accumulated and was deposited at the periphery of distorted ER‐derived PB‐I protein bodies. Synthesis of Aβ peptide in the ER lumen severely inhibited the synthesis and deposition of seed storage proteins, resulting in the generation of many small and abnormally appearing PB bodies. This ultrastructural change was accounted for by ER stress leading to the accumulation of aggregated Aβ peptide in the ER lumen and a coordinated increase in ER‐resident molecular chaperones such as BiPs and PDIs in Aβ‐expressing plants. Microarray analysis also confirmed that expression of several BiPs, PDIs and OsbZIP60 containing putative transmembrane domains was affected by the ER stress response. Aβ‐expressing transgenic rice kernels exhibited an opaque and shrunken phenotype. When grain phenotype and expression levels were compared among transgenic rice grains expressing several different recombinant peptides, such detrimental effects on grain phenotype were correlated with the expressed peptide causing ER stress rather than expression levels.     

β‐sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma

Short synthetic peptides homologous to the central region of Aβ but bearing proline residues as β‐sheet blockers have been shown in vitro to bind to Aβ with high affinity, partially inhibit Aβ fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood–brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAβ11, an 11‐residue β‐sheet breaker peptide that inhibits Aβ fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both ¹²⁵I‐YiAβ11 and ¹²⁵I‐PUT‐YiAβ11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all d ‐enantiomers of YiAβ11 and PUT‐YiAβ11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient × surface area product at the BBB was five‐ sevenfold higher in the cortex and hippocampus for the ¹²⁵I‐PUT‐d ‐YiAβ11 compared to the ¹²⁵I‐d ‐YiAβ11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT‐d ‐YiAβ11 retains its ability to partially inhibit Aβ fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five‐ to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine‐modified β‐sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 371–382, 1999     

RAC1mutation is not a predictive biomarker for PI3’‐kinase‐β‐selective pathway‐targeted therapy

Mutational activation of RAC1 is detected in ~7% of cutaneous melanoma, with the most frequent mutation (RAC1^C85T) encoding for RAC1^P29S. RAC1^P29S is a fast‐cycling GTPase that leads to accumulation of RAC1^P29S‐GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1^P29S propagates its pro‐tumorigenic effects remains unclear. RAC1‐GTP is reported to activate the beta isoform of PI3’‐kinase (PIK3CB/PI3Kβ) leading to downstream activation of PI3’‐lipid signaling. Hence, we employed both genetic and isoform‐selective pharmacological inhibitors to test if RAC1^P29S propagates its oncogenic signaling in melanoma through PI3Kβ. We observed that RAC1^P29S‐expressing melanoma cells were largely insensitive to inhibitors of PI3Kβ. Furthermore, RAC1^P29S melanoma cell lines showed variable sensitivity to pan‐class 1 (α/β/γ/δ) PI3’‐kinase inhibitors, suggesting that RAC1‐mutated melanoma cells may not rely on PI3’‐lipid signaling for their proliferation. Lastly, we observed that RAC1^P29S‐expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 → PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1‐mutated melanoma.