Photodynamic priming modulates cellular ATP levels to overcome P-glycoprotein-mediated drug efflux in chemoresistant triple-negative breast cancer.

P-glycoprotein (P-gp, ABCB1) is a well-researched ATP-binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P-gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non-toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub-cytotoxic photodynamic therapy process, can inhibit P-gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL-MDA-MB-231) and chemosensitive (MDA-MB-231) triple-negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P-gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL-MDA-MB-231 cells, but not in chemosensitive MDA-MB-231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P-gp in VBL-MDA-MB-231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P-gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Second-site suppressor mutations reveal connection between the drug-binding pocket and nucleotide-binding domain 1 of human P-glycoprotein (ABCB1).

Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4. This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.

Natural medicinal compounds target signal transduction pathways to overcome ABC drug efflux transporter-mediated multidrug resistance in cancer.

ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 are the major players in drug efflux-mediated multidrug resistance (MDR), which severely affects the efficacy of chemotherapy. Several synthetic compounds block the drug transport by ABC transporters; however, they exhibit a narrow therapeutic window, and produce side effects in non-target normal tissues. Conversely, the downregulation of the expression of ABC drug transporters seems to be a promising strategy to reverse MDR in cancer cells. Several signaling pathways, such as NF-κB, STAT3, Gli, NICD, YAP/TAZ, and Nrf2 upregulate the expression of ABC drug transporters in drug-resistant cancers. Recently, natural medicinal compounds have gained importance to overcome the ABC drug-efflux pump-mediated MDR in cancer. These compounds target transcription factors and the associated signal transduction pathways, thereby downregulating the expression of ABC transporters in drug-resistant cancer cells. Several potent natural compounds have been identified as lead candidates to synergistically enhance chemotherapeutic efficacy, and a few of them are already in clinical trials. Therefore, modulation of signal transduction pathways using natural medicinal compounds for the reversal of ABC drug transporter-mediated MDR in cancer is a novel approach for improving the efficiency of the existing chemotherapeutics. In this review, we discuss the modulatory role of natural medicinal compounds on cellular signaling pathways that regulate the expression of ABC transporters in drug-resistant cancer cells.

Advances in the structure, mechanism and targeting of chemoresistance-linked ABC transporters.

Cancer cells frequently display intrinsic or acquired resistance to chemically diverse anticancer drugs, limiting therapeutic success. Among the main mechanisms of this multidrug resistance is the overexpression of ATP-binding cassette (ABC) transporters that mediate drug efflux, and, specifically, ABCB1, ABCG2 and ABCC1 are known to cause cancer chemoresistance. High-resolution structures, biophysical and in silico studies have led to tremendous progress in understanding the mechanism of drug transport by these ABC transporters, and several promising therapies, including irradiation-based immune and thermal therapies, and nanomedicine have been used to overcome ABC transporter-mediated cancer chemoresistance. In this Review, we highlight the progress achieved in the past 5 years on the three transporters, ABCB1, ABCG2 and ABCC1, that are known to be of clinical importance. We address the molecular basis of their broad substrate specificity gleaned from structural information and discuss novel approaches to block the function of ABC transporters. Furthermore, genetic modification of ABC transporters by CRISPR-Cas9 and approaches to re-engineer amino acid sequences to change the direction of transport from efflux to import are briefly discussed. We suggest that current information regarding the structure, mechanism and regulation of ABC transporters should be used in clinical trials to improve the efficiency of chemotherapeutics for patients with cancer.

Giving a signal: how protein phosphorylation helps Bacillus navigate through different life stages.

Protein phosphorylation is a universal mechanism regulating a wide range of cellular responses across all domains of life. The antagonistic activities of kinases and phosphatases can orchestrate the life cycle of an organism. The availability of bacterial genome sequences, particularly Bacillus species, followed by proteomics and functional studies have aided in the identification of putative protein kinases and protein phosphatases, and their downstream substrates. Several studies have established the role of phosphorylation in different physiological states of Bacillus species as they pass through various life stages such as sporulation, germination, and biofilm formation. The most common phosphorylation sites in Bacillus proteins are histidine, aspartate, tyrosine, serine, threonine, and arginine residues. Protein phosphorylation can alter protein activity, structural conformation, and protein-protein interactions, ultimately affecting the downstream pathways. In this review, we summarize the knowledge available in the field of Bacillus signaling, with a focus on the role of protein phosphorylation in its physiological processes.

Anopheles gambiae mosGILT regulates innate immune genes and zpg expression.

Gene-edited mosquitoes lacking a g amma-interferon-inducible lysosomal thiol reductase-like protein, namely ( mosGILT null ) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILT null A. gambiae was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILT null A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg , an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILT null mosquitoes. These results provide the crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Specific mRNA lipid nanoparticles and acquired resistance to ticks.

Acquired resistance to ticks can develop when animals are repeatedly exposed to ticks. Recently, acquired resistance to Ixodes scapularis was induced in guinea pigs immunized with an mRNA-lipid nanoparticle vaccine (19ISP) encoding 19 I. scapularis proteins. Here, we evaluated specific mRNAs present in 19ISP to identify critical components associated with resistance to ticks. A lipid nanoparticle containing 12 mRNAs which included all the targets within 19ISP that elicited strong humoral responses in guinea pigs, was sufficient to induce robust resistance to ticks. Lipid nanoparticles containing fewer mRNAs or a single mRNA were not able to generate strong resistance to ticks. All lipid nanoparticles containing salp14 mRNA, however, were associated with increased redness at the tick bite site - which is the first manifestation of acquired resistance to ticks. This study demonstrates that more than one I. scapularis target within 19ISP is required for resistance to ticks, and that additional targets may also play a role in this process.

Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport.

P-glycoprotein (P-gp, ABCB1) transports structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs, thus contributing to multidrug-resistant cancer. Cryo-EM structures of human P-gp revealed that TMHs 4 and 10 contribute to the formation of the drug-binding cavity and undergo conformational changes during drug transport. To assess the role of the conformational changes in TMH4 and TMH10 during drug transport, we generated two mutants (TMH4-7A and TMH10-7A), each containing seven alanine substitutions. Analysis of the drug efflux function of these mutants using 15 fluorescent substrates revealed that most of the substrates were transported, indicating that even seven mutations in an individual helix have no significant effect on transport function. We then designed the TMH4,10-14A mutant combining seven mutations in both TMHs 4 and 10. Interestingly, when the TMH4,10-14A mutant was tested with 15 substrates, there was no efflux observed for fourteen. The basal ATPase activity of the TMH4,10-14A mutant, similar to that of the WT protein, was inhibited by zosuquidar but was not stimulated by verapamil or rhodamine 6G. Molecular dynamics simulations indicated that the mutations cause TMHs 4 and 10 to pack tighter to their proximal helices, reducing their independent mobility. In aggregate, our findings demonstrate the critical role of the residues of homologous TMHs 4 and 10 for substrate transport, consistent with conformational changes observed in the structure of P-gp.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

CD55 Facilitates Immune Evasion by Borrelia crocidurae, an Agent of Relapsing Fever.

Relapsing fever, caused by diverse Borrelia spirochetes, is prevalent in many parts of the world and causes significant morbidity and mortality. To investigate the pathoetiology of relapsing fever, we performed a high-throughput screen of Borrelia-binding host factors using a library of human extracellular and secretory proteins and identified CD55 as a novel host binding partner of Borrelia crocidurae and Borrelia persica, two agents of relapsing fever in Africa and Eurasia. CD55 is present on the surface of erythrocytes, carries the Cromer blood group antigens, and protects cells from complement-mediated lysis. Using flow cytometry, we confirmed that both human and murine CD55 bound to B. crocidurae and B. persica. Given the expression of CD55 on erythrocytes, we investigated the role of CD55 in pathological B. crocidurae-induced erythrocyte aggregation (rosettes), which enables spirochete immune evasion. We showed that rosette formation was partially dependent on host cell CD55 expression. Pharmacologically, soluble recombinant CD55 inhibited erythrocyte rosette formation. Finally, CD55-deficient mice infected with B. crocidurae had a lower pathogen load and elevated proinflammatory cytokine and complement factor C5a levels. In summary, our results indicate that CD55 is a host factor that is manipulated by the causative agents of relapsing fever for immune evasion. IMPORTANCE Borrelia species are causative agents of Lyme disease and relapsing fever infections in humans. B. crocidurae causes one of the most prevalent relapsing fever infections in parts of West Africa. In the endemic regions, B. crocidurae is present in ~17% of the ticks and ~11% of the rodents that serve as reservoirs. In Senegal, ~7% of patients with acute febrile illness were found to be infected with B. crocidurae. There is little information on host-pathogen interactions and how B. crocidurae manipulates host immunity. In this study, we used a high-throughput screen to identify host proteins that interact with relapsing fever-causing Borrelia species. We identified CD55 as one of the host proteins that bind to B. crocidurae and B. persica, the two causes of relapsing fever in Africa and Eurasia. We show that the interaction of B. crocidurae with CD55, present on the surface of erythrocytes, is key to immune evasion and successful infection in vivo. Our study further shows the role of CD55 in complement regulation, regulation of inflammatory cytokine levels, and innate immunity during relapsing fever infection. Overall, this study sheds light on host-pathogen interactions during relapsing fever infection in vivo.

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks.

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.

Immunomodulation by Mosquito Salivary Protein AgSAP Contributes to Early Host Infection by Plasmodium.

Malaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium-infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria. IMPORTANCE Malaria is a vector-borne disease caused by Plasmodium sporozoites. When an anopheline mosquito bites its host, it releases Plasmodium sporozoites as well as saliva components. Mosquito proteins have the potential to serve as antigens to prevent or influence malaria without directly targeting the pathogen. This may help set a new paradigm for vaccine development. In this study, we have elucidated the role of a novel salivary antigen, named Anopheles gambiae sporozoite-associated protein (AgSAP). The results presented here show that AgSAP interacts with Plasmodium falciparum and Plasmodium berghei sporozoites and modulates local inflammatory responses in the skin. Furthermore, our results show that AgSAP is a novel mosquito salivary antigen that influences the early stages of Plasmodium infection in the vertebrate host. Individuals living in countries where malaria is endemic generate antibodies against AgSAP, which indicates that AgSAP can serve as a biomarker for disease prevalence and epidemiological analysis.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies.

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

mRNA vaccination induces tick resistance and prevents transmission of the Lyme disease agent.

Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases, and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticle­containing nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recognition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection. Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially other tick-borne infections.

The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector.

Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of 'spirochete' bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply 'syringes' however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a 'complement 1q domain-contained' molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.

Stringent Response in Mycobacteria: From Biology to Therapeutic Potential.

Mycobacterium tuberculosis is a human pathogen that can thrive inside the host immune cells for several years and cause tuberculosis. This is due to the propensity of M. tuberculosis to synthesize a sturdy cell wall, shift metabolism and growth, secrete virulence factors to manipulate host immunity, and exhibit stringent response. These attributes help M. tuberculosis to manage the host response, and successfully establish and maintain an infection even under nutrient-deprived stress conditions for years. In this review, we will discuss the importance of mycobacterial stringent response under different stress conditions. The stringent response is mediated through small signaling molecules called alarmones "(pp)pGpp". The synthesis and degradation of these alarmones in mycobacteria are mediated by Rel protein, which is both (p)ppGpp synthetase and hydrolase. Rel is important for all central dogma processes-DNA replication, transcription, and translation-in addition to regulating virulence, drug resistance, and biofilm formation. Rel also plays an important role in the latent infection of M. tuberculosis. Here, we have discussed the literature on alarmones and Rel proteins in mycobacteria and highlight that (p)ppGpp-analogs and Rel inhibitors could be designed and used as antimycobacterial compounds against M. tuberculosis and non-tuberculous mycobacterial infections.

Structure-Activity Relationships of Pyrazolo[1,5-a]pyrimidin-7(4H)-ones as Antitubercular Agents.

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.

Role of post-translational modifications in the acquisition of drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the primary causes of deaths due to infectious diseases. The current TB regimen is long and complex, failing of which leads to relapse and/or the emergence of drug resistance. There is a critical need to understand the mechanisms of resistance development. With increasing drug pressure, Mycobacterium tuberculosis (Mtb) activates various pathways to counter drug-related toxicity. Signaling modules steer the evolution of Mtb to a variant that can survive, persist, adapt, and emerge as a form that is resistant to one or more drugs. Recent studies reveal that about 1/3rd of the annotated Mtb proteome is modified post-translationally, with a large number of these proteins being essential for mycobacterial survival. Post-translational modifications (PTMs) such as phosphorylation, acetylation, and pupylation play a salient role in mycobacterial virulence, pathogenesis, and metabolism. The role of many other PTMs is still emerging. Understanding the signaling pathways and PTMs may assist clinical strategies and drug development for Mtb. In this review, we explore the contribution of PTMs to mycobacterial physiology, describe the related cellular processes, and discuss how these processes are linked to drug resistance. A significant number of drug targets, InhA, RpoB, EmbR, and KatG, are modified at multiple residues via PTMs. A better understanding of drug-resistance regulons and associated PTMs will aid in developing effective drugs against TB.