Prediction of motor and non-motor Parkinson's disease symptoms using serum lipidomics and machine learning: a 2-year study.

Identifying biological factors which contribute to the clinical progression of heterogeneous motor and non-motor phenotypes in Parkinson's disease may help to better understand the disease process. Several lipid-related genetic risk factors for Parkinson's disease have been identified, and the serum lipid signature of Parkinson's disease patients is significantly distinguishable from controls. However, the extent to which lipid profiles are associated with clinical outcomes remains unclear. Untargeted high-performance liquid chromatography-tandem mass spectrometry identified >900 serum lipids in Parkinson's disease subjects at baseline (n = 122), and the potential for machine learning models using these lipids to predict motor and non-motor clinical scores after 2 years (n = 67) was assessed. Machine learning models performed best when baseline serum lipids were used to predict the 2-year future Unified Parkinson's disease rating scale part three (UPDRS III) and Geriatric Depression Scale scores (both normalised root mean square error = 0.7). Feature analysis of machine learning models indicated that species of lysophosphatidylethanolamine, phosphatidylcholine, platelet-activating factor, sphingomyelin, diacylglycerol and triacylglycerol were top predictors of both motor and non-motor scores. Serum lipids were overall more important predictors of clinical outcomes than subject sex, age and mutation status of the Parkinson's disease risk gene LRRK2. Furthermore, lipids were found to better predict clinical scales than a panel of 27 serum cytokines previously measured in this cohort (The Michael J. Fox Foundation LRRK2 Clinical Cohort Consortium). These results suggest that lipid changes may be associated with clinical phenotypes in Parkinson's disease.

Wollamide Cyclic Hexapeptides Synergize with Established and New Tuberculosis Antibiotics in Targeting Mycobacterium tuberculosis.

Shorter and more effective treatment regimens as well as new drugs are urgent priorities for reducing the immense global burden of tuberculosis (TB). As treatment of TB currently requires multiple antibiotics with diverse mechanisms of action, any new drug lead requires assessment of potential interactions with existing TB antibiotics. We previously described the discovery of wollamides, a new class of Streptomyces-derived cyclic hexapeptides with antimycobacterial activity. To further assess the value of the wollamide pharmacophore as an antimycobacterial lead, we determined wollamide interactions with first- and second-line TB antibiotics by determining fractional inhibitory combination index and zero interaction potency scores. In vitro two-way and multiway interaction analyses revealed that wollamide B1 synergizes with ethambutol, pretomanid, delamanid, and para-aminosalicylic acid in inhibiting the replication and promoting the killing of phylogenetically diverse clinical and reference strains of the Mycobacterium tuberculosis complex (MTBC). Wollamide B1 antimycobacterial activity was not compromised in multi- and extensively drug-resistant MTBC strains. Moreover, growth-inhibitory antimycobacterial activity of the combination of bedaquiline/pretomanid/linezolid was further enhanced by wollamide B1, and wollamide B1 did not compromise the antimycobacterial activity of the isoniazid/rifampicin/ethambutol combination. Collectively, these findings add new dimensions to the desirable characteristics of the wollamide pharmacophore as an antimycobacterial lead compound. IMPORTANCE Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur. Therefore, shorter, safer, more effective TB therapies are required, and these should ideally also be effective against drug-resistant strains of the bacteria that cause TB. This study shows that wollamide B1, a chemically optimized member of a new class of antibacterial compounds, inhibits the growth of drug-sensitive as well as multidrug-resistant Mycobacterium tuberculosis isolated from TB patients. In combination with TB antibiotics, wollamide B1 synergistically enhances the activity of several antibiotics, including complex drug combinations that are currently used for TB treatment. These new insights expand the catalogue of the desirable characteristics of wollamide B1 as an antimycobacterial lead compound that might inspire the development of improved TB treatments.

Distinct contributions of the innate immune receptors TLR2 and RP105 to formation and architecture of structured lung granulomas in mice infected with Mycobacterium tuberculosis.

Granulomas are key histopathological features of Mycobacterium tuberculosis (Mtb) infection, with complex roles in pathogen control and dissemination. Thus, understanding drivers and regulators of granuloma formation is important for improving tuberculosis diagnosis, treatment, and prevention. Yet, molecular mechanisms underpinning granuloma formation and dynamics remain poorly understood. Here we used low-dose Mtb infection of C57BL/6 mice, which elicits structured lung granulomas composed of central macrophage clusters encased by a lymphocyte mantle, alongside the disorganized lymphocyte and macrophage clusters commonly observed in Mtb-infected mice. Using gene-deficient mice, we observed that Toll-like receptor (TLR) 2 and the TLR-related Radioprotective 105 kDa protein (RP105) contributed to the extent and spatial positioning of pathology in infected lung tissues, consistent with functional cooperation between TLR2 and RP105 in the innate immune recognition of Mtb. In mice infected with the highly virulent Mtb clinical isolate HN878, TLR2, but not RP105, positively regulated the extent of central macrophage regions within structured granulomas. Moreover, RP105, but not TLR2, promoted the formation of structured lung granulomas, suggesting that the functions of RP105 as an innate immune sensor for Mtb reach beyond its roles as TLR2 co-receptor. TLR2 and RP105 contributions to lung pathology are governed by Mtb biology, as neither receptor affected the frequency or architecture of structured granulomas in mice infected with the reference strain Mtb H37Rv. Thus, by revealing distinctive as well as cooperative functions of TLR2 and RP105 in lung pathology, our data identify TLRs as molecular determinants of TB granuloma formation and architecture, and expand understanding of how interactions between innate immune receptors and Mtb shape TB disease manifestation.

Rab6b localizes to the Golgi complex in murine macrophages and promotes tumor necrosis factor release in response to mycobacterial infection.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

Microbiome-immune interactions in tuberculosis.

Tuberculosis (TB) remains an infectious disease of global significance and a leading cause of death in low- and middle-income countries. Significant effort has been directed towards understanding Mycobacterium tuberculosis genomics, virulence, and pathophysiology within the framework of Koch postulates. More recently, the advent of "-omics" approaches has broadened our appreciation of how "commensal" microbes have coevolved with their host and have a central role in shaping health and susceptibility to disease. It is now clear that there is a diverse repertoire of interactions between the microbiota and host immune responses that can either sustain or disrupt homeostasis. In the context of the global efforts to combatting TB, such findings and knowledge have raised important questions: Does microbiome composition indicate or determine susceptibility or resistance to M. tuberculosis infection? Is the development of active disease or latent infection upon M. tuberculosis exposure influenced by the microbiome? Does microbiome composition influence TB therapy outcome and risk of reinfection with M. tuberculosis? Can the microbiome be actively managed to reduce risk of M. tuberculosis infection or recurrence of TB? Here, we explore these questions with a particular focus on microbiome-immune interactions that may affect TB susceptibility, manifestation and progression, the long-term implications of anti-TB therapy, as well as the potential of the host microbiome as target for clinical manipulation.

Gut Microbial Signatures in Sporadic and Hereditary Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death and the third most common cancer in the world. Depending on the origin of the mutation, colorectal carcinomas are classified as sporadic or hereditary. Cancers derived from mutations appearing during life, affecting individual cells and their descendants, are called sporadic and account for almost 95% of the CRCs. Less than 5% of CRC cases result from constitutional mutations conferring a very high risk of developing cancer. Screening for hereditary-related cancers is offered to individuals at risk for hereditary CRC, who have either not undergone genetic evaluation or have uncertain genetic test results. In this review, we briefly summarize the main findings on the correlation between sporadic CRC and the gut microbiota, and we specifically focus on the few evidences about the role that gut microorganisms have on the development of CRC hereditary syndromes. The characterization of a gut microbiota associated with an increased risk of developing CRC could have a profound impact for prevention purposes. We also discuss the potential role of the gut microbiota as therapeutic treatment.

2-Aminooxazole as a Novel Privileged Scaffold in Antitubercular Medicinal Chemistry.

To obtain effective eradication of numerous infectious diseases such as tuberculosis, it is important to supply the medicinal chemistry arsenal with novel chemical agents. Isosterism and bioisosterism are widely known concepts in the field of early drug discovery, and in several cases, rational isosteric replacements have contributed to improved efficacy and physicochemical characteristics throughout the hit-to-lead optimization process. However, sometimes the synthesis of isosteres might not be as straightforward as that of the parent compounds, and therefore, novel synthetic strategies must be elaborated. In this regard, we herein report the evaluation of a series of N-substituted 4-phenyl-2-aminooxazoles that, despite being isosteres of a widely used nucleus such as the 2-aminothiazole, have been only seldom explored. After elaboration of a convenient synthetic strategy, a small set of 2-aminothiazoles and their 2-aminooxazole counterparts were compared with regard to antitubercular activity and physicochemical characteristics.

Rv0579 Is Involved in the Resistance to the TP053 Antitubercular Prodrug.

Tuberculosis remains one of the leading causes of death from a single pathogen globally. It is estimated that 1/4 of the world's population harbors latent tuberculosis, but only a 5-10% of patients will develop active disease. During latent infection, Mycobacterium tuberculosis can persist unaffected by drugs for years in a non-replicating state with low metabolic activity. The rate of the successful tuberculosis treatment is curbed by the presence of these non-replicating bacilli that can resuscitate after decades and also by the spread of M. tuberculosis drug-resistant strains. International agencies, including the World Health Organization, urge the international community to combat this global health emergency. The thienopyrimidine TP053 is a promising new antitubercular lead compound highly active against both replicating and non-replicating M. tuberculosis cells, with an in vitro MIC of 0.125 µg/ml. TP053 is a prodrug activated by the reduced form of the mycothiol-dependent reductase Mrx2, encoded by Rv2466c gene. After its activation, TP053 releases nitric oxide and a highly reactive metabolite, explaining its activity also against M. tuberculosis non-replicating cells. In this work, a new mechanism of TP053 resistance was discovered. M. tuberculosis spontaneous mutants resistant to TP053 were isolated harboring the mutation L240V in Rv0579, a protein with unknown function, but without mutation in Rv2466c gene. Recombineering method demonstrated that this mutation is linked to TP053 resistance. To better characterize Rv0579, the protein was recombinantly produced in Escherichia coli and a direct interaction between the Mrx2 activated TP053 and Rv0579 was shown by an innovative target-fishing experiment based on click chemistry. Thanks to achieved results, a possible contribution of Rv0579 in M. tuberculosis RNA metabolism was hypothesized, linked to toxin anti-toxin system. Overall, these data confirm the role of Rv0579 in TP053 resistance and consequently in the metabolism of this prodrug.

New Insights into the Mechanism of Action of the Thienopyrimidine Antitubercular Prodrug TP053.

The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and nonreplicating Mycobacterium tuberculosis (M. tuberculosis) cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. The investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against nonreplicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells as well as on nonreplicating cells. In summary, we explain the mechanism of action of TP053 on both replicating and nonreplicating M. tuberculosis and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting nonreplicating M. tuberculosis, which is shown to be killed by increased levels of nitric oxide.

Gut Microbiota Analysis in Postoperative Lynch Syndrome Patients.

Lynch syndrome (LS) is a dominantly inherited condition with incomplete penetrance, characterized by high predisposition to colorectal cancer (CRC), endometrial and ovarian cancers, as well as to other tumors. LS is associated with constitutive DNA mismatch repair (MMR) gene defects, and carriers of the same pathogenic variants can show great phenotypic heterogeneity in terms of cancer spectrum. In the last years, human gut microbiota got a foothold among risk factors responsible for the onset and evolution of sporadic CRC, but its possible involvement in the modulation of LS patients' phenotype still needs to be investigated. In this pilot study, we performed 16S rRNA gene sequencing of bacterial DNA extracted from fecal samples of 10 postoperative LS female patients who had developed colonic lesions (L-CRC) or gynecological cancers (L-GC). Our preliminary data show no differences between microbial communities of L-CRC and L-GC patients, but they plant the seed of the possible existence of a fecal microbiota pattern associated with LS genetic background, with Faecalibacterium prausnitzii, Parabacteroides distasonis, Ruminococcus bromii, Bacteroides plebeius, Bacteroides fragilis and Bacteroides uniformis species being the most significantly over-represented in LS patients (comprising both L-CRC and L-GC groups) compared to healthy subjects.

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase.

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 µg/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 µg/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 µg/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.

Shifts of Faecal Microbiota During Sporadic Colorectal Carcinogenesis.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.

Discovery and development of novel salicylate synthase (MbtI) furanic inhibitors as antitubercular agents.

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 µM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC99 = 156 µM), which is conceivably related to mycobactin biosynthesis inhibition.

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK.

Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.

Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity.

A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against Mycobacterium tuberculosis cell growth was evaluated. Some compounds such as phenolic hydrazine 1a and almost all heteroaromatic ones, especially 2, 5 and 7, are more active than isoniazid, and their activity against some M. tuberculosis MDR clinical isolates was determined. Compounds 1a and 7 present a selectivity index >1400 evaluated on MRC5 human fibroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell.

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 µM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

New prodrugs against tuberculosis.

The term 'prodrug' was first introduced by Albert in 1958. Generally, prodrugs can be utilized for improving active drug solubility and bioavailability, increasing drug permeability and absorption, modifying the distribution profile, preventing fast metabolism and excretion, and reducing toxicity. Previously, the prodrug approach was a final resort during the drug discovery process only after all other approaches had been exhausted. However, this strategy is now considered during the early stages of the drug development process. Most antitubercular agents are defined as 'prodrugs', including isoniazid and ethionamide. Thus, the prodrug approach could provide novel targets for the rational design of more effective treatments for tuberculosis (TB).

New and Old Hot Drug Targets in Tuberculosis.

Tuberculosis is an infectious disease caused by the bacillus Mycobacterium tuberculosis. The World Health Organization publishes global tuberculosis reports annually in order to provide the latest information in the surveillance of drug resistance. Given the alarming rise of resistance to antitubercular drugs worldwide, finding new cellular targets and developing new analogues or new compounds with greater potency against already known targets are both important aspects in fighting drug-sensitive and drug-resistant M. tuberculosis strains. In this context, the introduction of the phenotypic screens as an efficient tool for the identification of active compounds for tuberculosis drug discovery has improved the possibility to find new effective targets. With this review we describe the state of art of the currently well validated antitubercular drug targets as well as the advances in discovery of new ones. The main targets will be discussed starting from the oldest such as the enoyl reductase InhA which is constantly repurposed with new inhibitors, through the well assessed targets like the gyrase, the ATP synthetase or the RNA polymerase, up to the hot promiscuous targets decaprenylphosphoryl-Dribose oxidase DprE1 and the mycolic acid transporter MmpL3, or the newly validated and promising targets like the CTP synthetase.

Pyrrolidinone and pyrrolidine derivatives: Evaluation as inhibitors of InhA and Mycobacterium tuberculosis.

A series of GEQ analogues bearing pyrrolidinone or pyrrolidine cores were synthesized and evaluated against InhA, essential target for Mycobacterium tuberculosis (M.tb) survival. The compounds were also evaluated against M.tb H37Rv growth. Interestingly, some of the compounds, not efficient as InhA inhibitors, are active against M.tb with MICs up to 1.4 µM. In particular, compound 4b was screened with different M.tb mutated strains in order to identify the cellular target, but without success, suggesting a new possible mode of action.