Dual-drug RGD conjugates provide enhanced cytotoxicity to melanoma and non-small lung cancer cells

Y. Gilad, E. Noy, H. Senderowitz, A. Albeck, M. A. Firer, Gary Gellerman

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


To enhance the efficacy of targeted drug delivery, four new peptide-ligand conjugates were synthesized, each consisting of a cyclic RGDfK penta-peptide loaded with two anticancer drugs. The drug release profiles in different media of these new compounds and their cytotoxic activity against melanoma and non-small lung cancer cell lines were evaluated and compared with those of their singly loaded analogs. The cyclic RGDfK penta-peptide was selected as a targeting moiety because of its high affinity and selectivity to the αvβ3integrin receptor, which is frequently over-expressed in various types of cancer cells. The peptide's core was modified at the side chain of its Lys residue by coupling it with a sixth amino acid (AA) - either Lys (5a) or Ser (5b) (Lys/Ser splitter), resulting in two functional sites which enabled the loading of two therapeutic equivalents onto a single targeting carrier. Using Lys as a splitter resulted in two primary amines. Consequently, conjugates 1a and 1b were synthesized by coupling of 2 Chlorambucils (CLBs) or 2 Camptothecins (CPTs), respectively, to the primary amines of 5a. Conjugate 1c was synthesized from 5b by loading two equivalents of CLB on the amine and the hydroxyl of the Ser splitter, resulting in a homodimeric system with two distinct conjugation sites - amide and ester. The heterodimeric conjugate 1d of CLB and CPT was synthesized by loading each one of the primary amines of 5a with two different drugs - CLB and CPT. The doubling of drug equivalents loaded onto the targeting peptide correlated with enhanced cytotoxic efficacy of the conjugates towards cancer cells. The versatility of chemical linkages of the drugs to the peptides resulted in conjugates with different drug release profiles. Molecular dynamics simulations performed on conjugate 1d demonstrated that this compound occupies a conformational space similar to the bio-active conformation of an integrin-bound cyclic RGD peptide reference peptide (c(RGDf(NMe)V). The modified position in 1d (relative to the reference peptide) points away from the integrin, leading us to hypothesize that this peptide binds the integrin in a manner similar to that of the reference peptide thereby fulfilling a crucial requirement for targeted delivery. The strategy of dual drug loading on a single peptide carrier, gives rise to drugs with different mechanisms of action and release profiles, thus substantially increasing the efficacy of selective killing of tumor cells and while reducing the risk of the development of drug resistance.

Original languageEnglish
Pages (from-to)160-171
Number of pages12
Issue number2
StatePublished - 1 Mar 2016


  • RGD sequence
  • SPPS
  • drug release profiles
  • molecular dynamics simulations
  • multi-drug systems
  • peptide-drug conjugates
  • targeted drug delivery


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