Enhanced Cellular Uptake of a New, in Silico Identified Antitubercular Candidate by Peptide Conjugation Kata Horva ́ ti, † Bernadett Bacsa, † Nó ra Szabó , ‡ Sa ́ ndor Da ́ vid, †,‡ Ga ́ bor Mező , † Vince Grolmusz, §,# Bea ́ ta Ve ́ rtessy, ∥,⊥ Ferenc Hudecz, †,○ and Szilvia Bő sze* ,† † Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eö tvö s L. University, Budapest, Hungary ‡ Laboratory of Bacteriology, Kora ́ nyi National Institute for Tuberculosis and Respiratory Medicine, Budapest, Hungary § Protein Information Technology Group, Eö tvö s L. University, Budapest, Hungary ∥ Institute of Enzimology, Hungarian Academy of Science, Budapest, Hungary ⊥ Department of Applied Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary # Uratim Ltd., Budapest, Hungary ○ Department of Organic Chemistry, Eö tvö s L. University, Budapest, Hungary * S Supporting Information ABSTRACT: Mycobacterium tuberculosis is a successful patho- gen, and it can survive in infected macrophages in dormant phase for years and decades. The therapy of tuberculosis takes at least six months, and the slow-growing bacterium is resistant to many antibiotics. The development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies is urgently needed. In silico docking methods and structure-based drug design are useful bioinformatics tools for identifying new agents. A docking experiment to M. tuber- culosis dUTPase enzyme, which plays a key role in the bacterial metabolism, has resulted in 10 new antitubercular drug candidates. The uptake of antituberculars by infected macro- phages is limited by extracellular diffusion. The optimization of the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5 coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H 37 Rv culture in vitro; the chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5−OT20 tuftsin conjugate exhibited significant antimycobacterial activity on M. tuberculosis H 37 Rv infected MonoMac6 cells and inhibited intracellular bacteria. ■ INTRODUCTION It is estimated that more than one-third of the world’s population are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Latent TB is an asymptomatic phase of the disease during which bacilli do not multiply but persist within their host cells. Individuals with latent TB are assumed to harbor viable tubercle bacilli in their body. These bacilli have the potential to reactivate and cause disease. Around 10% of infected individuals will become sick with active TB during their lifetime. The risk of active TB is 20 to 40 times higher among patients living with HIV/AIDS, those with diabetes, cancer patients, organ transplant recipients, and those undergoing treatment for autoimmune diseases. 1 Among all TB cases, 5% are multidrug-resistant TB (MDR- TB), and in 2010, MDR-TB caused at least 150 000 deaths. MDR-TB strains are resistant to at least isoniazid and rifampicin, the two most effective first-line antituberculars. Additionally, a growing number of XDR-TB (extensively drug- resistant TB; resistant to isoniazid, rifampicin, any fluoroquino- lones, and any of the second-line anti-TB injectable drugs) was reported. 2 The therapy of resistant TB can take up to two years with drugs that are less effective, more expensive, and more toxic. The alarming number of bacterial strains resistant to current therapies led to the development of antituberculotics with novel mechanisms of action. M. tuberculosis dUTPase enzyme, which is required for mycobacterial growth, is one of the potential Received: April 30, 2011 Revised: January 19, 2012 Article pubs.acs.org/bc © XXXX American Chemical Society A dx.doi.org/10.1021/bc200221t | Bioconjugate Chem. XXXX, XXX, XXX−XXX