New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma.

Pharmacologically active salicylanilides (2-hydroxy-N-phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG]n, n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein.

Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH-Peptide Conjugates.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide-drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative-daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH2). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.

TXNL1 has dual functions as a redox active thioredoxin-like protein as well as an ATP- and redox-independent chaperone.

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity.

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Peptide Vaccines in Melanoma: Chemical Approaches towards Improved Immunotherapeutic Efficacy.

Cancer of the skin is by far the most common of all cancers. Although the incidence of melanoma is relatively low among skin cancers, it can account for a high number of skin cancer deaths. Since the start of deeper insight into the mechanisms of melanoma tumorigenesis and their strong interaction with the immune system, the development of new therapeutical strategies has been continuously rising. The high number of melanoma cell mutations provides a diverse set of antigens that the immune system can recognize and use to distinguish tumor cells from normal cells. Peptide-based synthetic anti-tumor vaccines are based on tumor antigens that elicit an immune response due to antigen-presenting cells (APCs). Although targeting APCs with peptide antigens is the most important assumption for vaccine development, peptide antigens alone are poorly immunogenic. The immunogenicity of peptide antigens can be improved not only by synthetic modifications but also by the assistance of adjuvants and/or delivery systems. The current review summarizes the different chemical approaches for the development of effective peptide-based vaccines for the immunotherapeutic treatment of advanced melanoma.

Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide-Drug Conjugates.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide-drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides.

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.

Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation.

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%-10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

[Application of Asn-Gly-Arg sequence based cyclic peptides for targeted tumor therapy]. / Asn-Gly-Arg szekvenciát tartalmazó ciklopeptidek alkalmazása a célzott tumorterápiában.

The in vivo antitumor effect of two NGR sequence containing peptide-daunomycin conjugates was studied on CD13+ Kaposi's sarcoma s.c. tumor model on SCID mice, and on orthotopically developed CD13- HT-29 colon adenocarcinoma SCID mouse model. Both tumor types were positive for integrins. Significant tumor growth inhibition was observed on both tumor types by the treatment with the conjugates (Dau=Aoa-GFLGK(cyclo[KNGRE]-GG)-NH2 (1) and Dau=Aoa-GFLGK(cyclo[NleNGRE]-GG)-NH2 (2)). KS conjugate 1 with rather stable construct was more potent in tumor growth inhibition that might be explained by the CD13 receptor recognition of NGR sequence. In contrast, conjugate 2 that has propensity to rearrange isoAsp derivative showed significantly higher inhibition on CD13- HT-29 tumor model that is related to the integrin binding of isoDGR sequence. Next to the low toxic side effect of the conjugates in comparison with the free daunomycin, the positive efficiency of the conjugates was detected by the lower proliferation index and lower neovascularization of the tumor tissue.

Cellular Internalization and Inhibition Capacity of New Anti-Glioma Peptide Conjugates: Physicochemical Characterization and Evaluation on Various Monolayer- and 3D-Spheroid-Based in Vitro Platforms.

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.

Old Polyanionic Drug Suramin Suppresses Detrimental Cytotoxicity of the Host Defense Peptide LL-37.

The host defense peptide LL-37 is the only human cathelicidin, characterized by pleiotropic activity ranging from immunological to anti-neoplastic functions. However, its overexpression has been associated with harmful inflammatory responses and apoptosis. Thus, for the latter cases, the development of strategies aiming to reduce LL-37 toxicity is highly desired as these have the potential to provide a viable solution. Here, we demonstrate that the reduction of LL-37 toxicity might be achieved by the impairment of its cell surface binding through interaction with small organic compounds that are able to alter the peptide conformation and minimize its cell penetration ability. In this regard, the performed cell viability and internalization studies showed a remarkable attenuation of LL-37 cytotoxicity toward colon and monocytic cells in the presence of the polysulfonated drug suramin. The mechanistic examinations of the molecular details indicated that this effect was coupled with the ability of suramin to alter LL-37 secondary structure via the formation of peptide-drug complexes. Moreover, a comparison with other therapeutic agents having common features unveiled the peculiar ability of suramin to optimize the binding to the peptide sequence. The newly discovered suramin action is hoped to inspire the elaboration of novel repurposing strategies aimed to reduce LL-37 cytotoxicity under pathological conditions.

Tailoring Uptake Efficacy of HSV-1 gD Derived Carrier Peptides.

Regions of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) were chosen to design carrier peptides based on the known tertiary structure of the virus entry receptor complexes. These complexes consist of the following: HSV-1 gD-nectin-1 and HSV-1 gD-herpesvirus entry mediator (HVEM). Three sets of peptides were synthesised with sequences covering the (i) N-terminal HVEM- and nectin-1 binding region -5-42, (ii) the 181-216 medium region containing nectin-1 binding sequences and (iii) the C-terminal nectin-1 binding region 214-255. The carrier candidates were prepared with acetylated and 5(6)-carboxyfluorescein labelled N-termini. The peptides were chemically characterised and their conformational features in solution were also determined. In vitro internalisation profile and intracellular localisation were evaluated on SH-SY5Y neuroblastoma cells. Peptide originated from the C-terminal region 224-247 of the HSV-1 gD showed remarkable internalisation compared to the other peptides with low to moderate entry. Electronic circular dichroism secondary structure studies of the peptides revealed that the most effectively internalised peptides exhibit high helical propensity at increasing TFE concentrations. We proved that oligopeptides derived from the nectin-1 binding region are promising candidates-with possibility of Lys237Arg and/or Trp241Phe substitutions-for side-reaction free conjugation of bioactive compounds-drugs or gene therapy agents-as cargos.

Drug Conjugation Induced Modulation of Structural and Membrane Interaction Features of Cationic Cell-Permeable Peptides.

Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.

Structure-Activity Relationship of HER2 Receptor Targeting Peptide and Its Derivatives in Targeted Tumor Therapy.

Human epidermal growth factor (HER2) is a transmembrane tyrosine kinase receptor that is frequently overexpressed in breast cancer. Its increased level prognoses a poor patient outcome and a high mortality rate. Despite the widening spectrum of therapies that are becoming available to treat HER2+ breast cancer, its side effects and resistance still make this protein a valuable object of research in targeted tumor therapy. The role of tumor-targeting peptides has become more and more prominent in the last few decades due to their simple synthesis and pharmakokinetic properties. Here, we examine two fluorescently-labeled HER2-specific peptides and their combined analogues that are developed to target the extracellular region of HER2. The peptides are investigated on breast cancer cell lines with different HER2 expression profiles. Moreover, their extracellular localization and specificity are confirmed by flow cytometry and confocal microscopy. Therefore, a new, combined HER2 binding conjugate is obtained that interacts with HER2-overexpressing cells with high affinity and specificity. Furthermore, secondary structure prediction reveals that the α-helical content of the peptides is associated with their receptor recognition. This highly specific conjugate can be used as a starting point for diagnostical or drug-targeting purposes in upcoming studies.

In Vivo Tumor Growth Inhibition and Antiangiogenic Effect of Cyclic NGR Peptide-Daunorubicin Conjugates Developed for Targeted Drug Delivery.

Among various homing devices, peptides containing the NGR tripeptide sequence represent a promising approach to selectively recognize CD13 receptor isoforms on the surface of tumor cells. They have been successfully used for the delivery of various chemotherapeutic drugs to tumor vessels. Here, we report on the murine plasma stability, in vitro and in vivo antitumor activity of our recently described bioconjugates containing daunorubicin as payload. Furthermore, CD13 expression of KS Kaposi's Sarcoma cell line and HT-29 human colon carcinoma cell line was investigated. Flow cytometry studies confirm the fast cellular uptake resulting in the rapid delivery of the active metabolite Dau = Aoa-Gly-OH to tumor cells. The increased in vitro antitumor effect might be explained by the faster rearrangement from NGR to isoDGR in case of conjugate 2 (Dau = Aoa-GFLGK(c[NleNGRE]-GG)-NH2) in comparison with conjugate 1 (Dau = Aoa-GFLGK(c[KNGRE]-GG)-NH2). Nevertheless, results indicated that both conjugates showed significant effect on inhibition of proliferation in the primary tumor and also on blood vessel formation making them a potential candidate for targeting angiogenesis processes in tumors where CD13 and integrins are involved.

[Optimization of peptide-drug conjugates for targeted tumor therapy]. / Célzott tumorterápiára alkalmas peptidhatóanyag konjugátumok szerkezetének optimalizálása.

In case of cancers with high mortality rate and lacking efficient medication there is a huge need of new, innovative treatments. Targeted tumor therapy, a real breakthrough in this field, is based on the concept that the antitumor agent is linked to a targeting molecule (e.g. peptide) specifically recognizing receptors or antigens that are tumor specific or overexpressed by tumor cells. The efficiency of this conjugate can be influenced by several factors. Among these, the structure of the targeting device, the type and number of the antitumor drug, its position in the conjugate and the chemical bonding of the drug to the targeting molecule are all important features that can determine receptor affinity and cellular uptake, and also the release and the cellular localization of the free drug or its active metabolite. Our goal in the framework of the grant NVKP_16-1-2016-0036 was to generate conjugates against cancers with high mortality rate. Through the below described studies, we introduce the course of the research process through which conjugates are optimized in order to develop more efficient drug candidates.

[Comparison of therapeutic peptides targeting pancreatic cancer]. / Hasnyálmirigytumort célzó terápiás irányítópeptidek összehasonlítása.

Despite the small number of cases, pancreatic cancer is one of the biggest challenges in tumor therapy as its treatment is not yet resolved and the expected 5-year survival rate is only 5%. Therefore, innovative solutions for pancreatic cancer are of great importance. Targeted tumor therapy might provide new possibilities in this field. In our research, we focused on finding peptide-based homing molecules and modified their structure to achieve better targeting properties. We compared several peptides that efficiently recognize receptors that are specific for or overexpressed by pancreatic cancer cells. Their structure-effect relationship was determined that can be useful during drug designing in the future. The antitumor effect of Dau=Aoa-GFLG-K(Dau=Aoa) SKAAKN-OH conjugate, which turned out to be the most efficient one during in vitro studies, were analyzed in vivo in female SCID mice. The obtained 30% inhibition, beside the low toxic side effects, might be a good starting point to develop further, more powerful conjugates.

Matrigel patterning reflects multicellular contractility.

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility.

A Convenient Synthetic Method to Improve Immunogenicity of Mycobacterium tuberculosis Related T-Cell Epitope Peptides.

Epitopes from different proteins expressed by Mycobacterium tuberculosis (Rv1886c, Rv0341, Rv3873) were selected based on previously reported antigenic properties. Relatively short linear T-cell epitope peptides generally have unordered structure, limited immunogenicity, and low in vivo stability. Therefore, they rely on proper formulation and on the addition of adjuvants. Here we report a convenient synthetic route to induce a more potent immune response by the formation of a trivalent conjugate in spatial arrangement. Chemical and structural characterization of the vaccine conjugates was followed by the study of cellular uptake and localization. Immune response was assayed by the measurement of splenocyte proliferation and cytokine production, while vaccine efficacy was studied in a murine model of tuberculosis. The conjugate showed higher tendency to fold and increased internalization rate into professional antigen presenting cells compared to free epitopes. Cellular uptake was further improved by the incorporation of a palmitoyl group to the conjugate and the resulted pal-A(P)I derivative possessed an internalization rate 10 times higher than the free epitope peptides. Vaccination of CB6F1 mice with free peptides resulted in low T-cell response. In contrast, significantly higher T-cell proliferation with prominent expression of IFN-γ, IL-2, and IL-10 cytokines was measured for the palmitoylated conjugate. Furthermore, the pal-A(P)I conjugate showed relevant vaccine efficacy against Mycobacterium tuberculosis infection.

Sequence modification of heptapeptide selected by phage display as homing device for HT-29 colon cancer cells to improve the anti-tumour activity of drug delivery systems.

Development of peptide-based conjugates for targeted tumour therapy is a current research topic providing new possibilities in cancer treatment. In this study, VHLGYAT heptapeptide selected by phage display technique for HT-29 human colon cancer was investigated as homing peptide for drug delivery. Daunomycin was conjugated to the N-terminus of the peptide directly or through Cathepsin B cleavable spacers. Conjugates showed moderate in vitro cytostatic effect. Therefore, sequence modifications were performed by Ala-scan and positional scanning resulting in conjugates with much higher bioactivity. Conjugates in which Gly was replaced by amino acids with bulky apolaric side chains provided the best efficacy. The influence of the cellular uptake, stability and drug release on the anti-tumour activity was investigated. It was found that mainly the difference in the cellular uptake of the conjugates generated the distinct effect on cell viability. One of the most efficient conjugate Dau = Aoa-LRRY-VHLFYAT-NH2 showed tumour growth inhibition on orthotopically developed HT-29 colon cancer in mice with negligible toxic side effect compared to the free drug. We also indicate that this sequence is not specific to HT-29 cells, but it has a remarkable effect on many other cancer cells. Nevertheless, the Phe-containing conjugate was more active in all cases compared to the conjugate with the parent sequence. The literature data suggested that this sequence is highly overlapped with peptides that recognize Hsp70 membrane bound protein overexpressed in many types of tumours.