Journal of Peptide Science J. Peptide Sci. 11: 37–44 (2005) Published online 7 July 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.587 Tuftsin-AZT conjugate: potential macrophage targeting for AIDS therapy MATI FRIDKIN, a * HAIM TSUBERY, a,b ESTHER TZEHOVAL, c AMI VONSOVER, ‡ LAURA BIONDI, d FERNANDO FILIRA d and RANIERO ROCCHI d a Department of Organic Chemistry, b Biological Chemistry and c Immunology, The Weizmann Institute of Science, Rehovot, Israel 76100 d Department of Organic Chemistry, University of Padova, Italy Received 15 March 04; Revised 22 April 04; Accepted 28 April 04 Abstract: The IgG-derived immunomodulating peptide tuftsin, Thr-Lys-Pro-Arg, is recognized by specific receptors on phagocytic cells, notably macrophages, and is capable of targeting proteins and peptides to these sites. Aiming to target 3 ′ -azido-3 ′ - deoxythymidine (AZT) to HIV-infected macrophages, a conjugate of AZT with tuftsin was synthesized. The AZT-tuftsin chimera possesses the characteristic capacities of its two components. Thus, like AZT, it inhibits reverse transcriptase activity and HIV- antigen expression, and similarly to tuftsin, it stimulates IL-1 release from mouse macrophages and augments the immunogenic function of the cells. Importantly, the conjugate is not cytotoxic to T-cells. The results suggest that the AZT-tuftsin conjugate might have potential use in AIDS therapy. Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd. Keywords: AZT; tuftsin; HIV; macrophages INTRODUCTION The inhibitor of viral reverse transcriptase 3 ′ -azido- 3 ′ -deoxythymidine (AZT) is one of the major drugs currently employed in AIDS therapy [1], affecting both enhanced survival and the quality of life in treated individuals. However, AZT application is accompanied by toxic side effects, particularly destruction of bone marrow cells that stems from its powerful cytotoxic nature [2]. Hence, administration of AZT, as well as other cytotoxic agents such as dideoxycytidine (ddC) or dideoxyinosine (ddI), directly to the human immunodeficiency virus (HIV)-infected cells is highly desirable. Decreasing the required drug doses and preventing or minimizing their action on non-infected cells would also reduce the harmful side effects. Potentially this objective may be achieved by covalent attachment of the therapeutically active agent of choice to a leading-targeting molecule, such as peptide or antibody, which is recognized by specific receptors of selected cells. Several attempts toward the realization of AZT-targeting were made, including its conjugation to the anti-transferin receptor antibody OX-26 for brain Abbreviations: AIDS, acquired immunodeficiency syndrome; AZT, 3 ′ - azido-3 ′ -deoxythymidine; DMEM, Dulbecco’s modified Eagle’s medium; HIV, human immunodeficiency virus; HPLC, high pressure liquid chromatography; PBS, phosphate buffer saline; PMC, 2,2,5,7,8- pentamethyl-chromane-6-sulfonyl; TLC, thin layer chromatography; WBC, white blood cells. * Correspondence to: Mati Fridkin, Department of Organic Chem- istry, The Weizmann Institute of Science, Rehovot, Israel 76100; e- mail: mati.fridkin@weizmann.ac.il ‡ Deceased targeting [3], its attachment to a 14-amino acid residue peptide for targeting toward the chemokine receptor on the T cell-line [4], or to a neoglycoprotein-director toward CD4 lymphocytes [5]. The immunoglobulin G associated phagocytosis- stimulating-peptide tuftsin, Thr-Lys-Pro-Arg, augments a wide spectrum of biological activities expressed by phagocytic cells, primarily macrophages, monocytes and neutrophils [6]. Following rather avid binding to specific receptors on these cells, i.e. binding constants of ∼10 −8 M [6–9], tuftsin notably enhances phagocyto- sis, cell motility, bactericidal and tumoricidal activities, immune response and the release of cytokines such as IL-1, IL-6 and TNF, indicating that the peptide may have significant and diverse clinical applications [6]. Initial studies on human cancer patients demon- strated that tuftsin is non-toxic and its administration leads to marked leukocytosis, i.e. elevation in white blood cells (WBC) and enhanced cytotoxicity of patients’ mononuclear cells toward tumor target cells [6,10,11]. Leukopenia, i.e. a reduction in WBC count, occurs in a large proportion of AIDS patients. This may be due to HIV affecting the bone marrow stem cells, as well as due to the application of substances causing myelo- toxic side effects [2]. With regard to clinical implications, the occurrence of leukopenia in AIDS patients means that certain therapies, e.g. chemotherapy of Kaposis sarcoma or lymphoma, or AZT therapy, should not be applied. Moreover, certain opportunistic infections are likely to be more frequent in patients with severe leukopenia than in those with normal or a mild reduc- tion in WBC. The effect of tuftsin on leukocytosis leading Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd.