Alternative Drug Targets Research Articles

Vitiligo: From mechanisms of disease to treatable pathways.

Vitiligo is a chronic autoimmune-mediated disease characterised by the loss of pigmentary melanocytes in the epidermis. Vitiligo is associated with loss of functional epithelium and significant reductions in quality of life with limited long-term treatment options, highlighting a continued unmet clinical need. A comprehensive understanding of the pathophysiology and newly investigated treatment pathways may guide multimodal treatment strategies and identify future drug targets. The pathology of vitiligo is multifactorial; however, environmental insults in genetically susceptible populations may lead to disease development. Autoreactive CD8+ T-cells that target melanocytes and release inflammatory mediators, including interferon-γ and interleukins 2, 6, 15, 17 and 33 among others, have been identified in vitiligo pathogenesis. Treatment modalities for vitiligo revolve around six broad disease concepts, including procedural modalities (tissue and cellular grafting), phototherapy, stem cells, anti-inflammatories, genetic polymorphisms and antioxidants/vitamins/herbals. Genetic polymorphisms, such as catalase gene variations and toll-like receptor polymorphisms, along with stem cell targets such as melanocytes derived from stem cells, have been implicated in vitiligo onset and possible treatment. Novel JAK-STAT inhibitors have been recently investigated for vitiligo, whereas topical corticosteroids and calcineurin inhibitors continue to be used. Vitamin D, vitamin E, zinc, copper, piperine, pseudo catalase and other vitamins/herbals may improve vitiligo outcomes primarily through antioxidant supplementation pathways. Future studies should investigate alternative drug pathways and targets implicated in vitiligo in large patient cohorts, as well as treatments that target suspected causative immune cells, including memory T-cells, which may provide long-lasting disease-free remission.

Druggable genome-wide Mendelian randomization for identifying the role of integrated stress response in therapeutic targets of bipolar disorder

For bipolar disorder (BD), the inconsistency of treatment guidelines and the long phases of pharmacological adjustment remain major challenges. BD is known to be comorbid with many medical and psychiatric conditions and they may share inflammatory and stress-related aetiologies, which could give rise to this association. The integrated stress response (ISR) responds to various stress conditions that lead to alterations in cellular homeostasis. However, as a causative mechanism underlying cognitive deficits and neurodegeneration in a broad range of brain disorders, the impact of ISR on BD is understudied. Mendelian randomization has been widely used to repurpose licensed drugs and discover novel therapeutic targets. Thus, we aimed to identify novel therapeutic targets for BD and analyze their pathophysiological mechanisms, using the summary data-based Mendelian Randomization (SMR) and Bayesian colocalization (COLOC) methods to integrate the summary-level data of the GWAS on BD and the expression quantitative trait locus (eQTL) study in blood. We utilized the GWAS data including 41,917 BD cases and 371,549 controls from the Psychiatric Genomics Consortium and the eQTL data from 31,684 participants of predominantly European ancestry from the eQTLGen consortium. The SMR analysis identified the EIF2B5 gene that was associated with BD due to no linkage but pleiotropy or causality. The COLOC analysis strongly suggested that EIF2B5 and the trait of BD were affected by shared causal variants, and thus were colocalized. Utilizing data in EpiGraphDB we find other putative causal BD genes (EIF2AK4 and GSK3B) to prioritize potential alternative drug targets.

Alzheimer's disease and its treatment-yesterday, today, and tomorrow.

Alois Alzheimer described the first patient with Alzheimer's disease (AD) in 1907 and today AD is the most frequently diagnosed of dementias. AD is a multi-factorial neurodegenerative disorder with familial, life style and comorbidity influences impacting a global population of more than 47 million with a projected escalation by 2050 to exceed 130 million. In the USA the AD demographic encompasses approximately six million individuals, expected to increase to surpass 13 million by 2050, and the antecedent phase of AD, recognized as mild cognitive impairment (MCI), involves nearly 12 million individuals. The economic outlay for the management of AD and AD-related cognitive decline is estimated at approximately 355 billion USD. In addition, the intensifying prevalence of AD cases in countries with modest to intermediate income countries further enhances the urgency for more therapeutically and cost-effective treatments and for improving the quality of life for patients and their families. This narrative review evaluates the pathophysiological basis of AD with an initial focus on the therapeutic efficacy and limitations of the existing drugs that provide symptomatic relief: acetylcholinesterase inhibitors (AChEI) donepezil, galantamine, rivastigmine, and the N-methyl-D-aspartate receptor (NMDA) receptor allosteric modulator, memantine. The hypothesis that amyloid-β (Aβ) and tau are appropriate targets for drugs and have the potential to halt the progress of AD is critically analyzed with a particular focus on clinical trial data with anti-Aβ monoclonal antibodies (MABs), namely, aducanumab, lecanemab and donanemab. This review challenges the dogma that targeting Aβ will benefit the majority of subjects with AD that the anti-Aβ MABs are unlikely to be the "magic bullet". A comparison of the benefits and disadvantages of the different classes of drugs forms the basis for determining new directions for research and alternative drug targets that are undergoing pre-clinical and clinical assessments. In addition, we discuss and stress the importance of the treatment of the co-morbidities, including hypertension, diabetes, obesity and depression that are known to increase the risk of developing AD.

Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

Sildenafil, alone and in combination with imipramine or escitalopram, display antidepressant-like effects in an adrenocorticotropic hormone-induced (ACTH) rodent model of treatment-resistant depression

BackgroundMajor depressive disorder (MDD) represents a challenge with high prevalence and limited effectiveness of existing treatments, particularly in cases of treatment-resistant depression (TRD). Innovative strategies and alternative drug targets are therefore necessary. Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, is known to exert neuroplastic, anti-inflammatory, and antioxidant properties, and is a promising antidepressant drug candidate. AimTo investigate whether sildenafil monotherapy or in combination with a known antidepressant, can elicit antidepressant-like effects in an adrenocorticotropic hormone (ACTH)-induced rodent model of TRD. MethodsACTH-naïve and ACTH-treated male Sprague-Dawley (SD) rats received various sub-acute drug treatments, followed by behavioural tests and biochemical analyses conversant with antidepressant actions. ResultsSub-chronic ACTH treatment induced significant depressive-like behaviour in rats, evidenced by increased immobility during the forced swim test (FST). Sub-acute sildenafil (10 mg/kg) (SIL-10) (but not SIL-3), and combinations of imipramine (15 mg/kg) (IMI-15) and sildenafil (3 mg/kg) (SIL-3) or escitalopram (15 mg/kg) (ESC-15) and SIL-3, exhibited significant antidepressant-like effects. ACTH treatment significantly elevated hippocampal levels of brain-derived neurotrophic factor (BDNF), serotonin, norepinephrine, kynurenic acid (KYNUA), quinolinic acid (QUINA), and glutathione. The various mono- and combined treatments significantly reversed some of these changes, whereas IMI-15 + SIL-10 significantly increased glutathione disulfide levels. ESC-15 + SIL-3 significantly reduced plasma corticosterone levels. ConclusionThis study suggests that sildenafil shows promise as a treatment for TRD, either as a stand-alone therapy or in combination with a traditional antidepressant. The neurobiological mechanism underlying the antidepressant-like effects of the different sildenafil mono- and combination therapies reflects a multimodal action and cannot be explained in full by changes in the individually measured biomarker levels.

Core-genome-mediated promising alternative drug and multi-epitope vaccine targets prioritization against infectious Clostridium difficile.

Prevention of Clostridium difficile infection is challenging worldwide owing to its high morbidity and mortality rates. C. difficile is currently being classified as an urgent threat by the CDC. Devising a new therapeutic strategy become indispensable against C. difficile infection due to its high rates of reinfection and increasing antimicrobial resistance. The current study is based on core proteome data of C. difficile to identify promising vaccine and drug candidates. Immunoinformatics and vaccinomics approaches were employed to construct multi-epitope-based chimeric vaccine constructs from top-ranked T- and B-cell epitopes. The efficacy of the designed vaccine was assessed by immunological analysis, immune receptor binding potential and immune simulation analyses. Additionally, subtractive proteomics and druggability analyses prioritized several promising and alternative drug targets against C. difficile. These include FMN-dependent nitroreductase which was prioritized for pharmacophore-based virtual screening of druggable molecule databases to predict potent inhibitors. A MolPort-001-785-965 druggable molecule was found to exhibit significant binding affinity with the conserved residues of FMN-dependent nitroreductase. The experimental validation of the therapeutic targets prioritized in the current study may worthy to identify new strategies to combat the drug-resistant C. difficile infection.

Phosphoproteomics of patient-derived xenografts identifies targets and markers associated with sensitivity and resistance to EGFR blockade in colorectal cancer.

Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.

Astrocytic CD44 Deficiency Reduces the Severity of Kainate-Induced Epilepsy.

Epilepsy affects millions of people worldwide, yet we still lack a successful treatment for all epileptic patients. Most of the available drugs modulate neuronal activity. Astrocytes, the most abundant cells in the brain, may constitute alternative drug targets. A robust expansion of astrocytic cell bodies and processes occurs after seizures. Highly expressed in astrocytes, CD44 adhesion protein is upregulated during injury and is suggested to be one of the most important proteins associated with epilepsy. It connects the astrocytic cytoskeleton to hyaluronan in the extracellular matrix, influencing both structural and functional aspects of brain plasticity. Herein, we used transgenic mice with an astrocyte CD44 knockout to evaluate the impact of the hippocampal CD44 absence on the development of epileptogenesis and ultrastructural changes at the tripartite synapse. We demonstrated that local, virally-induced CD44 deficiency in hippocampal astrocytes reduces reactive astrogliosis and decreases the progression of kainic acid-induced epileptogenesis. We also observed that CD44 deficiency resulted in structural changes evident in a higher dendritic spine number along with a lower percentage of astrocyte-synapse contacts, and decreased post-synaptic density size in the hippocampal molecular layer of the dentate gyrus. Overall, our study indicates that CD44 signaling may be important for astrocytic coverage of synapses in the hippocampus and that alterations of astrocytes translate to functional changes in the pathology of epilepsy.

Interactions of IDO and the Kynurenine Pathway with Cell Transduction Systems and Metabolism at the Inflammation-Cancer Interface.

The mechanisms underlying a relationship between inflammation and cancer are unclear, but much emphasis has been placed on the role of tryptophan metabolism to kynurenine and downstream metabolites, as these make a substantial contribution to the regulation of immune tolerance and susceptibility to cancer. The proposed link is supported by the induction of tryptophan metabolism by indoleamine-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO), in response to injury, infection or stress. This review will summarize the kynurenine pathway and will then focus on the bi-directional interactions with other transduction pathways and cancer-related factors. The kynurenine pathway can interact with and modify activity in many other transduction systems, potentially generating an extended web of effects other than the direct effects of kynurenine and its metabolites. Conversely, the pharmacological targeting of those other systems could greatly enhance the efficacy of changes in the kynurenine pathway. Indeed, manipulating those interacting pathways could affect inflammatory status and tumor development indirectly via the kynurenine pathway, while pharmacological modulation of the kynurenine pathway could indirectly influence anti-cancer protection. While current efforts are progressing to account for the failure of selective IDO1 inhibitors to inhibit tumor growth and to devise means of circumventing the issue, it is clear that there are wider factors involving the relationship between kynurenines and cancer that merit detailed consideration as alternative drug targets.

Elucidating the pH gating mechanism of Helicobacter pyloris urea channel UreI.

Over 50% of the world population suffer from chronic gastric infection with Helicobacter pyloris which is linked to peptic ulcer disease and stomach cancer. Yet, the efficacy of the common therapy including antibiotics is decreasing. An alternative drug target could be H. pyloris pH gatedinner-membrane urea channel UreI, which is pivotal for the survival of the pathogen in the acidic environment of the human stomach. However, despite in vivo studies and high-resolution structures in the open and closed state, HpUreIs gating mechanism is still elusive. Using yeast complementation assays, we tested (i) homologues urea channels, (ii) point mutations of charged residues also on the cytoplasmic side, and (iii) variants carrying changes at the periplasmic side at the N- and C-terminus as well as in periplasmic loop 1 (PL1). The functionality and pH gating behaviour of these constructs are compared to the wild type protein in the physiological relevant pH range from 4.0 to 7.0. Our in vivo studies are guided and accompanied by an extensive sequence- and structure-based analysis of homologues urea channels. The results question the hypothesis of PL1 and PL2 constituting the main pH-sensor of HpUreI but confirm the importance of the N- and C-terminus in the pH gating mechanism. For example, additional amino acids at the C-terminus provoke a urea impermeable HpUreI, emphasizing its importance for the stability of the open state. Furthermore, the urea channels from Helicobacter hepaticus and Streptococcus salivarius, lacking such extensive periplasmic loops, show similar pH dependence as HpUreI. Overall, yeast complementation assays are an cost-effective tool for testing qualitative differences in protein variant activity for a broad range of solutes.

Comparative Proteomics and Genome-Wide Druggability Analyses Prioritized Promising Therapeutic Targets against Drug-Resistant Leishmania tropica

Leishmania tropica is a tropical parasite causing cutaneous leishmaniasis (CL) in humans. Leishmaniasis is a serious public health threat, affecting an estimated 350 million people in 98 countries. The global rise in antileishmanial drug resistance has triggered the need to explore novel therapeutic strategies against this parasite. In the present study, we utilized the recently available multidrug resistant L. tropica strain proteome data repository to identify alternative therapeutic drug targets based on comparative subtractive proteomic and druggability analyses. Additionally, small drug-like compounds were scanned against novel targets based on virtual screening and ADME profiling. The analysis unveiled 496 essential cellular proteins of L. tropica that were nonhomologous to the human proteome set. The druggability analyses prioritized nine parasite-specific druggable proteins essential for the parasite's basic cellular survival, growth, and virulence. These prioritized proteins were identified to have appropriate binding pockets to anchor small drug-like compounds. Among these, UDPase and PCNA were prioritized as the top-ranked druggable proteins. The pharmacophore-based virtual screening and ADME profiling predicted MolPort-000-730-162 and MolPort-020-232-354 as the top hit drug-like compounds from the Pharmit resource to inhibit L. tropica UDPase and PCNA, respectively. The alternative drug targets and drug-like molecules predicted in the current study lay the groundwork for developing novel antileishmanial therapies.

Non-kinase targeting of oncogenic c-Jun N-terminal kinase (JNK) signaling: the future of clinically viable cancer treatments.

c-Jun N-terminal Kinases (JNKs) have been identified as key disease drivers in a number of pathophysiological settings and central oncogenic signaling nodes in various cancers. Their roles in driving primary tumor growth, positively regulating cancer stem cell populations, promoting invasion and facilitating metastatic outgrowth have led JNKs to be considered attractive targets for anti-cancer therapies. However, the homeostatic, apoptotic and tumor-suppressive activities of JNK proteins limit the use of direct JNK inhibitors in a clinical setting. In this review, we will provide an overview of the different JNK targeting strategies developed to date, which include various ATP-competitive, non-kinase and substrate-competitive inhibitors. We aim to summarize their distinct mechanisms of action, review some of the insights they have provided regarding JNK-targeting in cancer, and outline the limitations as well as challenges of all strategies that target JNKs directly. Furthermore, we will highlight alternate drug targets within JNK signaling complexes, including recently identified scaffold proteins, and discuss how these findings may open up novel therapeutic options for targeting discrete oncogenic JNK signaling complexes in specific cancer settings.

Insights from the molecular docking analysis of compounds from Vitex negundoi with targets from Klebsiella pneumoniai causing urinary tract infection.

Antimicrobial resistance among bacterial strains has emerged out to be a serious threat and contributes to the loss of effectiveness of the common antibiotics. New Delhi metallo-β-lactamase-1 (blaNDM-1) is an enzyme present in several pathogenic bacteria with a high incidence in Klebsiella pneumoniaie and plays a crucial role in the development of antibacterial resistance. Mur enzymes are also important alternative drug targets in addition to blaNDM-1 which are crucial for the survival of the bacteria. Vitex negundoi is an aromatic medicinaltree with proven antibacterial properties. Fifteen compounds from V. negundo were evaluated for their inhibitory effects on the target proteins blaNDM-1, Mur C, Mur E and Mur F of K. pneumoniae through molecular docking using the Glide (xp) module of Schrodinger. ADME toxicity was also predicted for all the fifteen compounds in the QikProp module. The docking results revealed that the compounds agnuside, negundoside and isoorientin showed promising inhibitory effects on all four targets blaNDM-1, Mur C, Mur E and Mur F of K. pneumoniae with docking scores greater than -7 kcal/mol and reasonable hydrogen bond interactions. The findings of this study provide a lead for developing novel drugs against potent multidrug-resistant K. pneumoniae.

Indoleamine 2,3-dioxygenase 2 immunohistochemical expression in medullary thyroid carcinoma: implications in prognosis and immunomodulatory effects

BackgroundThe linkage between IDO2 expression and cancer progression is still unclear, particularly in medullary thyroid carcinoma (MTC). Our purpose is to unveil the potential correlations between IDO2 status, clinical-pathological parameters, patients’ prognosis, and the possible immunomodulatory functions in MTC.MethodsImmunohistochemical expression levels of IDO2 were evaluated in the resected MTC surgical specimens and corresponding lymph nodes. CD4 + T cell infiltration was also evaluated by immunohistochemical analysis in the MTC tissues. The association of the IDO2 expression level with clinicopathologic characteristics, overall survival (OS)/recurrence-free survival (RFS), and CD4 + T cell infiltration were retrospectively investigated.ResultsHigh expression of IDO2 is closely associated with more aggressive clinicopathological features, such as multifocality, ETE, a higher pT stage and especially a higher pN stage. Moreover, a significant difference in RFS was observed between the IDO2-high and IDO2-low groups. IDO2 expression of lymph node tissues was significantly related to the metastasis status. Furthermore, we found that IDO2 expression is negatively correlated with CD4 + T cell infiltrations in MTC tissues.ConclusionThe expression level of IDO2 is associated with aggressive characteristics and is predictive of poor prognosis in patients with MTC. Also, an interesting observation is that IDO2 involvement in MTC showed a moderate sexual dimorphism, of which female patients tend to be more affected by IDO2 status. Moreover, our results showed the potential immunomodulatory functions of IDO2. The close relationship between IDO2 and CD4 + T cell infiltration in the MTC microenvironment, together with its potential prognostic implications, makes it possible for IDO2 to serve as an alternative drug target in cancer immunotherapy and as a new prognostic tool.

Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells.

Cell cycle-related kinase (CCRK) is most closely related to cyclin-dependent protein kinase, which may activate cyclin-dependent kinase 2 and is associated with the growth of human cancer cells. However, the expression and function of CCRK in the pathogenesis of clear cell renal cell cancer (ccRCC) are unclear. Herein, this research aimed to explore the potential mechanism of the targeted regulation of CCRK by miR-335-5p on the proliferation and tumorigenicity of human ccRCC cells. The results showed that CCRK was significantly overexpressed in ccRCC tissues and cells, and knockdown of the CCRK expression by shRNA inhibited cell proliferation in vitro and in vivo and enhanced cell apoptosis in vitro, which indicated that CCRK could be a potential target for antitumour drugs in the treatment of ccRCC. Moreover, miR-335-5p was found to bind directly to the 3′ untranslated region of CCRK, was expressed at markedly low levels in ccRCC cells, and was closely associated with the tumour stage. The overexpression of CCRK partially reversed the inhibitory effects of miR-335-5p on the cell growth of ccRCC, which implied that miR-335-5p could serve as a promising tumour inhibitor for ccRCC. In summary, CCRK could serve as an alternative antitumour drug target, and miR-335-5p could be a promising therapeutic tumour inhibitor for ccRCC treatment.

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues.

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing.

Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as Cne and Cga. Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 μg/mL against the wild-type Cne-H99 (Cne-WT or Cne). In contrast, CMN inhibited the intein-less mutant strain (Cne-Mut) with a 16-fold higher MIC. Interestingly, Aspergillus fumigatus and a few Candida species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in Cne. CMN also inhibited Cne intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC50 of CMN was 4.6 μg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating Cne cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat Cne and possibly Cga infections.

Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target.

Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world’s population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical ‘BioC-BioH(3)’ paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

Molecular mechanisms governing aquaporin relocalisation.

The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.