Protein-ligand complex for structure-based design: impact on the affinity and antitumor activity of new tubulin ligands

Resumen del trabajo presentado en el XVIII Congreso de la Sociedad Española de Química Terapéutica, celebrado en Salamanca (España), del 23 al 26 de enero de 2018Microtubules, made of ¿ß¿tubulin heterodimers, are the key components of the cytoskeleton and play a crucial role in many cellular processes, such as cell motility, morphogenesis and mitosis.[1] Interference with microtubule dynamics induces cell cycle arrest during mitosis and triggers cell death. Compounds that interact with tubulin, especially those binding at the colchicine domain, have been deeply investigated as anticancer drugs due to their dual mechanism of action as antimitotics and as vascular disrupting agents.[2,3] Our research group has recently described a new family of colchicine¿domain binders, based on a cyclohexanedione skeleton, with potent antiproliferative activity against tumor and endothelial cells.[4] Moreover, to gain insight into the binding mode of these cyclohexanediones, we have determined the crystal structure of ¿ß¿tubulin in complex with our hit compound (TUB075). Based on this detailed information and by applying the affinity maps program cGRILL, a structurebased synthesis of new cyclohexanedione derivatives has been accomplished with the objective of improving their affinity for tubulin and their antitumor activity. Following this approach, we have obtained new compounds with potent antiproliferative activity against tumor and endothelial cells (IC50=8¿31 nM) and with the highest Kb value reported for compounds binding at the colchicine site in tubulin. Additional studies have shown that they arrest cell cycle at G2/M and disrupt a network of endothelial cells. Moreover they keep antiproliferative activity against cell lines overexpressing P¿gp, further supporting the potential of these compounds.The financial support of the Spanish MINECO (SAF2012‐39760‐C02‐01 and SAF 2015‐64629‐C2‐1‐R), Comunidad de Madrid (BIPEDD2; ref P2010/BMD‐2457) and the COST action CM1407 (to M J. P.P., S.L., M.O.S. and J.F.D.) is sincerely acknowledge

Derivados de nucleósidos para el tratamiento de infecciones por Leishmania

Derivados de nucleósidos para el tratamiento de infecciones por Leishmania. Compuestos derivados de nucleósidos y su uso para el tratamiento de enfermedades infecciosas causadas por Leishmania. Además la invención se refiere a las composiciones farmacéuticas que contienen dichos compuestos.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de AlcaláB1 Patente sin examen previ

Nucleósidos modificados para el tratamiento de infecciones por Leishmania

Nucleósidos modificados para el tratamiento de infecciones por Leishmania. Compuestos derivados de nucleósidos y su uso para el tratamiento de enfermedades infecciosas causadas por Leishmania. Además la invención se refiere a las composiciones farmacéuticas que contienen dichos compuestos.Consejo Superior de Investigaciones Científicas (España), Universidad de AlcaláA1 Solicitud de patente con informe sobre el estado de la técnic

Recent Advances in Thymidine Kinase 2 (TK2) Inhibitors and New Perspectives for Potential Applications

Thymidine kinase 2 (TK2), encoded on chromosome 16q22 of the human genome, is a deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of the pyrimidine deoxynucleosides 2’-deoxythymidine (dThd), 2’-deoxyuridine (dUrd) and 2’- deoxycytidine (dCyd) to the corresponding deoxynucleoside 5’-monophosphate derivatives. In contrast to the S-phase-specific thymidine kinase 1 (TK1), TK2 is constitutively expressed in the mitochondria and plays an important role in providing dNTPs for the replication and maintenance of mitochondrial DNA (mtDNA). The severe mitochondrial DNA depletion syndrome (MDS) has been associated with mutations in TK2, resulting in mtDNA depletion, isolated skeletal myopathy, and death of the individual at an early stage of life. Some antiviral nucleoside analogs, such as 3’-azido-dThd (AZT) that is targeting the human immunodeficiency virus (HIV)-encoded reverse transcriptase, are substrates for TK2 and it has been proposed that the mitochondrial toxicity observed after prolonged treatment with such drugs could be due to their interaction with TK2. Therefore, the design of specific TK2 inhibitors may be useful to investigate the role of TK2 in the maintenance and homeostasis of mitochondrial dNTP pools and its contribution to the mitochondrial toxicity of several antiviral and anticancer drugs. Since 2000, several potent and selective TK2 inhibitors have been described. Besides bringing together previously reported inhibitors, special attention will be paid in this review to the new families of TK2 inhibitors more recently described, together with modeling studies and biological assays. Moreover, the last section will be focused on several recent investigations that suggest that depletion of mtDNA can take place both in tumorigenesis and during cancer treatment with certain nucleoside analogues.Peer reviewe

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule >crystallization chaperone> 5′-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5′-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5′-O-tritylinosine on human TP. A cavity in which 5′-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (Vmax/Km of mutant enzyme ∼50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5′-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP. © 2009 Elsevier Inc. All rights reserved.Peer Reviewe

Leishmania infantum EndoG is an endo/exo-nuclease essential for parasite survival

EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5′ exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites. © 2014 Rico et al.Peer Reviewe

Structure, physiological role, and specific inhibitors of human thymidine kinase 2 (TK2): Present and future

Human mitochondrial thymidine kinase (TK2) is a pyrimidine deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of pyrimidine deoxynucleosides to their corresponding deoxynucleoside 5′-monophosphates by γ-phosphoryl transfer from ATP. In resting cells, TK2 is suggested to play a key role in the mitochondrial salvage pathway to provide pyrimidine nucleotides for mitochondrial DNA (mtDNA) synthesis and maintenance. However, recently the physiological role of TK2 turned out to have direct clinical relevance as well. Point mutations in the gene encoding TK2 have been correlated to mtDNA disorders in a heterogeneous group of patients suffering from the so-called mtDNA depletion syndrome (MDS). TK2 activity could also be involved in mitochondrial toxicity associated to prolonged treatment with antiviral nucleoside analogues like AZT and FIAU. Therefore, TK2 inhibitors can be considered as valuable tools to unravel the role of TK2 in the maintenance and homeostasis of mitochondrial nucleotide pools and mtDNA, and to clarify the contribution of TK2 activity to mitochondrial toxicity of certain antivirale. Highly selective TK-2 inhibitors having an acyclic nucleoside structure and efficiently discriminating between TK-2 and the closely related TK-1 have already been reported. It is actually unclear whether these agents efficiently reach the inner mitochondrial compartment. In the present review article, structural features of TK2, MDS-related mutations observed in TK2 and their role in MDS will be discussed. Also, an update on novel and selective TK2 inhibitors will be provided. © 2008 Wiley Periodicals, Inc.Peer Reviewe

Exploring acyclic nucleoside analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)-butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display Ki values of 0.42 and 0.27 μM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.Peer Reviewe