Spotlights
Trends in Microbiology April 2014, Vol. 22, No. 4
Spectinamides: a challenge, a proof, and a suggestion Miguel Viveiros1 and Marco Pieroni2 1
Grupo de Micobacte´rias, Unidade de Microbiologia Me´dica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal 2 Dipartimento di Farmacia, Centro Interdipartimentale BIOPHARMANET_TEC, Universita` di Parma, Parco Area delle Scienze 27, 43124 Parma, Italy
New drugs and shorter regimens are needed to fight resistant forms of tuberculosis. Screening of chemical libraries for mining hit compounds is the common approach to find new antituberculars. A new medicinal chemistry and biology directed research rational has recently been successfully described by Lee and coworkers in Nature Medicine. Lee and co-workers have recently published a paper in which the basic principles of medicinal chemistry and bacterial physiology have come together to produce a noticeable piece of science [1]. Mycobacterium tuberculosis is known to have peculiar characteristics, such as a remarkable infectiveness for humans, capacity to elude the immune response and latency, which results in a timeless public health challenge to control and eradicate. Being one of the oldest human pathogens, it has developed countless adaptive survival strategies to overcome the immune response and, in the past 70 years, antimycobacterial therapy [2]. As such, 3608 research on antimycobacterials must deal with these peculiarities. The structure-based approach for the design of novel antituberculars also represents a challenge: research hints have made it apparent that a whole-cell phenotypic high-throughput screening (HTS) approach is more likely to yield lead candidates for further advancement than the isolated target approach [3,4]. For instance, bedaquiline and PA824, two of the most promising new antitubercular agents, are the result of the improvement of hit compounds identified after a random antitubercular screening of inhouse chemical libraries at Johnson & Johnson and CibaGeigy, respectively [5]. Although debating on what is the best drug discovery approach is beyond the aim of this Spotlight, it must be recognized that the phenotypic susceptibility assays give a quick and fairly reliable feedback on the therapeutic potential of a molecule, for which penetration into the bacterial cell and growth inhibition are promptly assessed. On the contrary, sometimes a potent inhibitor of a vital bacterial target fails to show any ‘cidal’ effect because of the impossibility to reach the intended target, either because it cannot penetrate or because once inside the cell it is readily extruded [6]. And this feature is even more Corresponding author: Viveiros, M. ([email protected]). 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.008
170
significant in the case of the greasy and thick mycobacterial cell wall. In the work by Lee and colleagues, the spectinamide derivatives were rationally designed upon computational aid focused on a phenotypic trait of M. tuberculosis: the efflux-mediated resistance to spectinomycin [1]. The homology model of the M. tuberculosis rRNA 16S helix 34 spectinomycin binding site was built based on the crystal structure of that of Escherichia coli in complex with spectinomycin. The model gave hints on why spectinomycin itself is inactive toward M. tuberculosis, and how to make it an effective antitubercular. In particular, it appeared clear how heterocyclic appendages could be attached through an amide linker at the C-3 position of the spectinomycin core, therefore affording extra ribosomal contacts with residues R24, R25, F31 and G1193. Moreover, the intramolecular interactions generated by the heterocyclic nitrogen and the amide, decreased the polarity of the molecules that, as such, are less susceptible to efflux. The efflux activity is crucial for bacterial survival and persistence under antimicrobial stress [7,8]. The authors address the issue of extrusion by assessing the activity of the compounds in knockout mutants for Rv1258c, one of the best characterized mycobacterial efflux pumps and responsible for efflux of spectinomycin. Proof is provided that the structure of the molecule determines its affinity for the efflux systems. Indeed, the findings show that decreasing the affinity for the transporter enzyme warrants lower MICs (i.e., increased antimicrobial activity by intracellular retention). However, if one looks at the other side of the coin, the same results can be obtained if this efflux is inhibited; a suggestion that can be taken into consideration, as many data report that this is a valuable strategy to overcome intrinsic resistance in M. tuberculosis [8,9]. This novel and unexplored strategy for the rational design of new drugs and the planning of therapeutic regimens is particularly promising to prevent acquired multidrug resistance in tuberculosis as treatment consists of a cocktail of drugs to be taken for, at least, six months. The majority of research on antituberculars has been focused on finding more active growth inhibitors, assuming they reach their bacterial target. On the contrary, this work suggests an innovative path to follow that is based on inhibition of efflux, rather than the increase of the potency. The clear evidence presented that inhibiting active efflux increased the bactericidal activity of spectinomycin derivatives opens a plethora of opportunities for innovative and effective combinatory therapeutic regimens, where antimicrobials
Spotlights can tackle M. tuberculosis inside the macrophages where persistence starts. Efflux pumps, such as the Rv1258c, are known to promote not only antimicrobial extrusion but also intra-macrophage survival of M. tuberculosis and escape killing by lysosomal agents [7,10]. This peculiar host–parasite relational feature of efflux pumps should be the focus of more effective drugs and regimens. The two most effective antitubercular agents known, isoniazid and rifampicin, have also been shown to be subject to intrinsic efflux in M. tuberculosis and therefore they can have their efficacy enhanced by concerted molecular and biological strategies to reduce active efflux and enhance macrophage killing activity [7,10]. In particular, the activity noticed under in vitro hypoxic conditions gives a reasonable idea of the potential of spectinamides against latent infection and should be further explored. This hypothesis was greatly favoured by the in vivo results obtained in this work, with excellent antitubercular activity observed in both acute and chronic tuberculosis models, comparing molecules that only differ from each other by their ability to inhibit efflux of one particular M. tuberculosis pump. As the study by Lee and colleagues elegantly demonstrates, compounds designed to avoid pump-mediated efflux will certainly have superior activity against hard-to-kill, drug-tolerant bacteria, leading to better in vivo efficacies and improved antitubercular regimens [1,7,10].
Trends in Microbiology April 2014, Vol. 22, No. 4
References 1 Lee, R.E. et al. (2014) Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux. Nat. Med. 20, 152–158 2 Comas, I. et al. (2013) Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45, 1176–1182 3 Pieroni, M. et al. (2011) Pyrido[1,2-a]benzimidazole-based agents active against tuberculosis (TB), multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB. ChemMedChem 6, 334–342 4 Onajole, O.K. et al. (2013) Preliminary structure–activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. J. Med. Chem. 56, 4093–4103 5 Zumla, A. et al. (2013) Advances in the development of new tuberculosis drugs and treatment regimens. Nat. Rev. Drug Discov. 12, 388–404 6 Swinney, D.C. and Anthony, J. (2011) How were new medicines discovered? Nat. Rev. Drug Discov. 10, 507–519 7 Viveiros, M. et al. (2012) Inhibitors of mycobacterial efflux pumps as potential boosters for anti-tubercular drugs. Expert Rev. Anti Infect. Ther. 10, 983–998 8 Balganesh, M. et al. (2012) Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates. Antimicrob. Agents Chemother. 56, 2643–2651 9 Machado, D. et al. (2012) Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis. PLoS ONE 7, e34538 10 Adams, K.N. et al. (2011) Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145, 39–53
Loop de loop: viral RNA evades IFIT1 targeting Justin A. Roby*, Brian D. Clarke*, and Alexander A. Khromykh Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, St Luca, Brisbane, Queensland 4072, Australia
In a landmark finding published in Science, Hyde et al. have demonstrated that a hairpin RNA structure adjacent to the 50 cap of alphavirus genomic RNA confers the ability of these viruses to evade restriction by the interferon-induced host protein IFIT1. The methylation at the 20 -O position of eukaryotic mRNAs serves to prevent recognition and translational-inhibition of host mRNA by the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1); an important mediator of the innate immune response [1]. The positive strand RNA viruses within the Alphavirus genus are important pathogens of humans that replicate in the cytoplasm and notably lack 20 -O methylation at the 50 untranslated region (UTR) of their genomic and subgenomic RNAs (gRNA and sgRNA) [2], thus exposing their RNAs to inhibition by Corresponding author: Khromykh, A.A. ([email protected]). * These authors contributed equally. 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.014
IFIT1. Despite this, alphaviruses are able to replicate in cells producing IFIT proteins upon stimulation with interferon (IFN) [3,4]. The means by which these viruses evade IFIT1 activity has only recently been revealed [3]. In order to investigate the mechanism that alphaviruses use to evade IFIT1 activity, Hyde et al. [3] have neatly exploited the availability of two Venezuelan equine encephalitis virus (VEEV) strains (parental and descendent); previously characterized for differential sensitivity to IFN treatment [5]. The attenuated VEEV strain TC83 encodes a G3A mutation within the 50 UTR that was associated with increased sensitivity to IFN [5]. The mechanisms responsible for this phenotype had not been elucidated despite evidence that this mutation leads to a structural rearrangement of the viral RNA [6]. In their recent article in Science, Hyde et al. identified host IFIT1 as the agent responsible for A3-VEEV IFN-mediated attenuation (Figure 1) [3]. The authors showed that the G3A mutation destabilizes a stem-loop at the beginning of the 50 UTR and exposes the non 20 -O methylated 50 cap to recognition by host IFIT1. Using HeLa cells transduced with short hairpin RNAs (shRNAs) targeting IFIT1 and knockout mice deficient in 171
Trends in Microbiology April 2014, Vol. 22, No. 4
Spectinamides: a challenge, a proof, and a suggestion Miguel Viveiros1 and Marco Pieroni2 1
Grupo de Micobacte´rias, Unidade de Microbiologia Me´dica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal 2 Dipartimento di Farmacia, Centro Interdipartimentale BIOPHARMANET_TEC, Universita` di Parma, Parco Area delle Scienze 27, 43124 Parma, Italy
New drugs and shorter regimens are needed to fight resistant forms of tuberculosis. Screening of chemical libraries for mining hit compounds is the common approach to find new antituberculars. A new medicinal chemistry and biology directed research rational has recently been successfully described by Lee and coworkers in Nature Medicine. Lee and co-workers have recently published a paper in which the basic principles of medicinal chemistry and bacterial physiology have come together to produce a noticeable piece of science [1]. Mycobacterium tuberculosis is known to have peculiar characteristics, such as a remarkable infectiveness for humans, capacity to elude the immune response and latency, which results in a timeless public health challenge to control and eradicate. Being one of the oldest human pathogens, it has developed countless adaptive survival strategies to overcome the immune response and, in the past 70 years, antimycobacterial therapy [2]. As such, 3608 research on antimycobacterials must deal with these peculiarities. The structure-based approach for the design of novel antituberculars also represents a challenge: research hints have made it apparent that a whole-cell phenotypic high-throughput screening (HTS) approach is more likely to yield lead candidates for further advancement than the isolated target approach [3,4]. For instance, bedaquiline and PA824, two of the most promising new antitubercular agents, are the result of the improvement of hit compounds identified after a random antitubercular screening of inhouse chemical libraries at Johnson & Johnson and CibaGeigy, respectively [5]. Although debating on what is the best drug discovery approach is beyond the aim of this Spotlight, it must be recognized that the phenotypic susceptibility assays give a quick and fairly reliable feedback on the therapeutic potential of a molecule, for which penetration into the bacterial cell and growth inhibition are promptly assessed. On the contrary, sometimes a potent inhibitor of a vital bacterial target fails to show any ‘cidal’ effect because of the impossibility to reach the intended target, either because it cannot penetrate or because once inside the cell it is readily extruded [6]. And this feature is even more Corresponding author: Viveiros, M. ([email protected]). 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.008
170
significant in the case of the greasy and thick mycobacterial cell wall. In the work by Lee and colleagues, the spectinamide derivatives were rationally designed upon computational aid focused on a phenotypic trait of M. tuberculosis: the efflux-mediated resistance to spectinomycin [1]. The homology model of the M. tuberculosis rRNA 16S helix 34 spectinomycin binding site was built based on the crystal structure of that of Escherichia coli in complex with spectinomycin. The model gave hints on why spectinomycin itself is inactive toward M. tuberculosis, and how to make it an effective antitubercular. In particular, it appeared clear how heterocyclic appendages could be attached through an amide linker at the C-3 position of the spectinomycin core, therefore affording extra ribosomal contacts with residues R24, R25, F31 and G1193. Moreover, the intramolecular interactions generated by the heterocyclic nitrogen and the amide, decreased the polarity of the molecules that, as such, are less susceptible to efflux. The efflux activity is crucial for bacterial survival and persistence under antimicrobial stress [7,8]. The authors address the issue of extrusion by assessing the activity of the compounds in knockout mutants for Rv1258c, one of the best characterized mycobacterial efflux pumps and responsible for efflux of spectinomycin. Proof is provided that the structure of the molecule determines its affinity for the efflux systems. Indeed, the findings show that decreasing the affinity for the transporter enzyme warrants lower MICs (i.e., increased antimicrobial activity by intracellular retention). However, if one looks at the other side of the coin, the same results can be obtained if this efflux is inhibited; a suggestion that can be taken into consideration, as many data report that this is a valuable strategy to overcome intrinsic resistance in M. tuberculosis [8,9]. This novel and unexplored strategy for the rational design of new drugs and the planning of therapeutic regimens is particularly promising to prevent acquired multidrug resistance in tuberculosis as treatment consists of a cocktail of drugs to be taken for, at least, six months. The majority of research on antituberculars has been focused on finding more active growth inhibitors, assuming they reach their bacterial target. On the contrary, this work suggests an innovative path to follow that is based on inhibition of efflux, rather than the increase of the potency. The clear evidence presented that inhibiting active efflux increased the bactericidal activity of spectinomycin derivatives opens a plethora of opportunities for innovative and effective combinatory therapeutic regimens, where antimicrobials
Spotlights can tackle M. tuberculosis inside the macrophages where persistence starts. Efflux pumps, such as the Rv1258c, are known to promote not only antimicrobial extrusion but also intra-macrophage survival of M. tuberculosis and escape killing by lysosomal agents [7,10]. This peculiar host–parasite relational feature of efflux pumps should be the focus of more effective drugs and regimens. The two most effective antitubercular agents known, isoniazid and rifampicin, have also been shown to be subject to intrinsic efflux in M. tuberculosis and therefore they can have their efficacy enhanced by concerted molecular and biological strategies to reduce active efflux and enhance macrophage killing activity [7,10]. In particular, the activity noticed under in vitro hypoxic conditions gives a reasonable idea of the potential of spectinamides against latent infection and should be further explored. This hypothesis was greatly favoured by the in vivo results obtained in this work, with excellent antitubercular activity observed in both acute and chronic tuberculosis models, comparing molecules that only differ from each other by their ability to inhibit efflux of one particular M. tuberculosis pump. As the study by Lee and colleagues elegantly demonstrates, compounds designed to avoid pump-mediated efflux will certainly have superior activity against hard-to-kill, drug-tolerant bacteria, leading to better in vivo efficacies and improved antitubercular regimens [1,7,10].
Trends in Microbiology April 2014, Vol. 22, No. 4
References 1 Lee, R.E. et al. (2014) Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux. Nat. Med. 20, 152–158 2 Comas, I. et al. (2013) Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45, 1176–1182 3 Pieroni, M. et al. (2011) Pyrido[1,2-a]benzimidazole-based agents active against tuberculosis (TB), multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB. ChemMedChem 6, 334–342 4 Onajole, O.K. et al. (2013) Preliminary structure–activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. J. Med. Chem. 56, 4093–4103 5 Zumla, A. et al. (2013) Advances in the development of new tuberculosis drugs and treatment regimens. Nat. Rev. Drug Discov. 12, 388–404 6 Swinney, D.C. and Anthony, J. (2011) How were new medicines discovered? Nat. Rev. Drug Discov. 10, 507–519 7 Viveiros, M. et al. (2012) Inhibitors of mycobacterial efflux pumps as potential boosters for anti-tubercular drugs. Expert Rev. Anti Infect. Ther. 10, 983–998 8 Balganesh, M. et al. (2012) Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates. Antimicrob. Agents Chemother. 56, 2643–2651 9 Machado, D. et al. (2012) Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis. PLoS ONE 7, e34538 10 Adams, K.N. et al. (2011) Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145, 39–53
Loop de loop: viral RNA evades IFIT1 targeting Justin A. Roby*, Brian D. Clarke*, and Alexander A. Khromykh Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, St Luca, Brisbane, Queensland 4072, Australia
In a landmark finding published in Science, Hyde et al. have demonstrated that a hairpin RNA structure adjacent to the 50 cap of alphavirus genomic RNA confers the ability of these viruses to evade restriction by the interferon-induced host protein IFIT1. The methylation at the 20 -O position of eukaryotic mRNAs serves to prevent recognition and translational-inhibition of host mRNA by the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1); an important mediator of the innate immune response [1]. The positive strand RNA viruses within the Alphavirus genus are important pathogens of humans that replicate in the cytoplasm and notably lack 20 -O methylation at the 50 untranslated region (UTR) of their genomic and subgenomic RNAs (gRNA and sgRNA) [2], thus exposing their RNAs to inhibition by Corresponding author: Khromykh, A.A. ([email protected]). * These authors contributed equally. 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.014
IFIT1. Despite this, alphaviruses are able to replicate in cells producing IFIT proteins upon stimulation with interferon (IFN) [3,4]. The means by which these viruses evade IFIT1 activity has only recently been revealed [3]. In order to investigate the mechanism that alphaviruses use to evade IFIT1 activity, Hyde et al. [3] have neatly exploited the availability of two Venezuelan equine encephalitis virus (VEEV) strains (parental and descendent); previously characterized for differential sensitivity to IFN treatment [5]. The attenuated VEEV strain TC83 encodes a G3A mutation within the 50 UTR that was associated with increased sensitivity to IFN [5]. The mechanisms responsible for this phenotype had not been elucidated despite evidence that this mutation leads to a structural rearrangement of the viral RNA [6]. In their recent article in Science, Hyde et al. identified host IFIT1 as the agent responsible for A3-VEEV IFN-mediated attenuation (Figure 1) [3]. The authors showed that the G3A mutation destabilizes a stem-loop at the beginning of the 50 UTR and exposes the non 20 -O methylated 50 cap to recognition by host IFIT1. Using HeLa cells transduced with short hairpin RNAs (shRNAs) targeting IFIT1 and knockout mice deficient in 171
