A consensus reverse docking approach for identification of a competitive inhibitor of acetyltransferase enhanced intracellular survival protein from Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.

Inhibition of Fosfomycin Resistance Protein FosB from Gram-Positive Pathogens by Phosphonoformate.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico-based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosBSa) and Bacillus cereus (FosBBc). In addition, we have determined a crystal structure of FosBBc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.

Discovery and Mechanistic Analysis of Structurally Diverse Inhibitors of Acetyltransferase Eis among FDA-Approved Drugs.

Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis-proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.

Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives.

Chloramphenicol nitroreductase (CNR), a drug-modifying enzyme from Haemophilus influenzae, has been shown to be responsible for the conversion of the nitro group into an amine in the antibiotic chloramphenicol (CAM). Since CAM structurally bears a 4-nitrobenzene moiety, we explored the substrate promiscuity of CNR by investigating its nitroreduction of 4-nitrobenzyl derivatives. We tested twenty compounds containing a nitrobenzene core, two nitropyridines, one compound with a vinylogous nitro group, and two aliphatic nitro compounds. In addition, we also synthesized twenty-eight 4-nitrobenzyl derivatives with ether, ester, and thioether substituents and assessed the relative activity of CNR in their presence. We found several of these compounds to be modified by CNR, with the enzyme activity ranging from 1 to 150% when compared to CAM. This data provides insights into two areas: (i) chemoenzymatic reduction of select compounds to avoid harsh chemicals and heavy metals routinely used in reductions of nitro groups and (ii) functional groups that would aid CAM in overcoming the activity of this enzyme.

Acetylation by Eis and Deacetylation by Rv1151c of Mycobacterium tuberculosis HupB: Biochemical and Structural Insight.

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.

Using MbtH-Like Proteins to Alter the Substrate Profile of a Nonribosomal Peptide Adenylation Enzyme.

MbtH-like proteins (MLPs) are required for soluble expression and/or optimal activity of some adenylation (A) domains of nonribosomal peptide synthetases. Because A domains can interact with noncognate MLP partners, how the function of an A domain, TioK, involved in the biosynthesis of the bisintercalator thiocoraline, is altered by noncognate MLPs has been investigated. Measuring TioK activity with 12 different MLPs from a variety of bacterial species by using a radiometric assay suggested that the A domain substrate promiscuity could be altered by foreign MLPs. Kinetic studies and bioinformatics analysis expanded the complexity of MLP functions and interactions.

Multifunctional Donepezil Analogues as Cholinesterase and BACE1 Inhibitors.

A series of 22 donepezil analogues were synthesized through alkylation/benzylation and compared to donepezil and its 6-O-desmethyl adduct. All the compounds were found to be potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), two enzymes responsible for the hydrolysis of the neurotransmitter acetylcholine in Alzheimer's disease patient brains. Many of them displayed lower inhibitory concentrations of EeAChE (IC50 = 0.016 ± 0.001 µM to 0.23 ± 0.03 µM) and EfBChE (IC50 = 0.11 ± 0.01 µM to 1.3 ± 0.2 µM) than donepezil. One of the better compounds was tested against HsAChE and was found to be even more active than donepezil and inhibited HsAChE better than EeAChE. The analogues with the aromatic substituents were generally more potent than the ones with aliphatic substituents. Five of the analogues also inhibited the action of ß-secretase (BACE1) enzyme.

Differential Effects of Linkers on the Activity of Amphiphilic Tobramycin Antifungals.

As the threat associated with fungal infections continues to rise and the availability of antifungal drugs remains a concern, it becomes obvious that the need to bolster the antifungal armamentarium is urgent. Building from our previous findings of tobramycin (TOB) derivatives with antifungal activity, we further investigate the effects of various linkers on the biological activity of these aminoglycosides. Herein, we analyze how thioether, sulfone, triazole, amide, and ether functionalities affect the antifungal activity of alkylated TOB derivatives against 22 Candida, Cryptococcus, and Aspergillus species. We also evaluate their impact on the hemolysis of murine erythrocytes and the cytotoxicity against mammalian cell lines. While the triazole linker appears to confer optimal activity overall, all of the linkers incorporated into the TOB derivatives resulted in compounds that are very effective against the Cryptococcus neoformans species, with MIC values ranging from 0.48 to 3.9 µg/mL.

Bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones: New classes of antibacterial/antifungal agents.

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones for their antibacterial and antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi. We investigated their potential to develop resistance against both bacteria and fungi by a multi-step resistance-selection method, explored their potential to induce the production of reactive oxygen species, and assessed their toxicity. In summary, we found that these compounds exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains with minimum inhibitory concentration (MIC) values ranging from <0.5 to >500µM against bacteria and 1.0 to >31.3µg/mL against fungi; and in most cases, they exhibited either superior or similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also observed minimal emergence of drug resistance to these newly synthesized compounds by bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the human Ether-à-go-go-related gene (hERG) channel with acceptable IC50 values ranging from 1.12 to 3.29µM. Overall, these studies show that bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial and antifungal agents.

The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

A biocatalytic approach to capuramycin analogues by exploiting a substrate permissive N-transacylase CapW.

Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from a methyl ester precursor as an acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed that this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.

Development of ebsulfur analogues as potent antibacterials against methicillin-resistant Staphylococcus aureus.

Antibiotic resistance is a worldwide problem that needs to be addressed. Staphylococcus aureus is one of the dangerous "ESKAPE" pathogens that rapidly evolve and evade many current FDA-approved antibiotics. Thus, there is an urgent need for new anti-MRSA compounds. Ebselen (also known as 2-phenyl-1,2-benzisoselenazol-3(2H)-one) has shown promising activity in clinical trials for cerebral ischemia, bipolar disorder, and noise-induced hearing loss. Recently, there has been a renewed interest in exploring the antibacterial properties of ebselen. In this study, we synthesized an ebselen-inspired library of 33 compounds where the selenium atom has been replaced by sulfur (ebsulfur derivatives) and evaluated them against a panel of drug-sensitive and drug-resistant S. aureus and non-S. aureus strains. Within our library, we identified three outstanding analogues with potent activity against all S. aureus strains tested (MIC values mostly ⩽2µg/mL), and numerous additional ones with overall very good to good antibacterial activity (1-7.8µg/mL). We also characterized the time-kill analysis, anti-biofilm ability, hemolytic activity, mammalian cytotoxicity, membrane-disruption ability, and reactive oxygen species (ROS) production of some of these analogues.

Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

Inhibition of glioma progression by a newly discovered CD38 inhibitor.

Glioma, the most common cancer of the central nervous system, has very poor prognosis and no effective treatment. It has been shown that activated microglia/macrophages in the glioma tumor microenvironment support progression. Hence, inhibition of the supporting effect of these cells may constitute a useful therapeutic approach. Recently, using a syngeneic mouse glioma progression model, we showed that the ectoenzyme CD38 regulated microglia activation and, in addition, that the loss of CD38 from the tumor microenvironment attenuated glioma progression and prolonged the life span of the tumor-bearing mice. These studies, which employed wild-type (WT) and Cd38(-/-) C57BL/6J mice, suggest that inhibition of CD38 in glioma microenvironment may be used as a new therapeutic approach to treat glioma. Our study tested this hypothesis. Initially, we found that the natural anthranoid, 4,5-dihydroxyanthraquinone-2-carboxylic acid (rhein), and its highly water-soluble tri-potassium salt form (K-rhein) are inhibitors of CD38 enzymatic (nicotinamide adenine dinucleotide glycohydrolase) activity (IC50 = 1.24 and 0.84 µM, respectively, for recombinant mouse CD38). Treatment of WT, but not Cd38(-/-) microglia with rhein and K-rhein inhibited microglia activation features known to be regulated by CD38 (lipopolysaccharide/IFN-γ-induced activation, induced cell death and NO production). Furthermore, nasal administration of K-rhein into WT, but not Cd38(-/-) C57BL/6J, mice intracranially injected with GL261 cells substantially and significantly inhibited glioma progression. Hence, these results serve as a proof of concept, demonstrating that targeting CD38 at the tumor microenvironment by small-molecule inhibitors of CD38, for example, K-rhein, may serve as a useful therapeutic approach to treat glioma.

Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers.

The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

Inhibition of aminoglycoside acetyltransferase resistance enzymes by metal salts.

Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn(2+) metal ions displayed an inhibitory effect on the resistance enzyme AAC(6')-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg(2+), Cr(3+), Cr(6+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Au(3+) with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2')-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6')-Ib', AAC(6')-Ie, AAC(6')-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection.

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents.

In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studies. Cytotoxicity was evaluated in mammalian cell lines. The fungal membrane-disruptive action of these analogues was studied by using the membrane-impermeable dye propidium iodide. TOB analogues bearing a linear alkyl chain at their 6″-position in a thioether linkage exhibited chain length-dependent antifungal activities. Analogues with C12 and C14 chains showed promising antifungal activities against tested fungal strains, with MIC values ranging from 1.95 to 62.5 mg/liter and 1.95 to 7.8 mg/liter, respectively. However, C4, C6, and C8 TOB analogues and TOB itself exhibited little to no antifungal activity. Fifty percent inhibitory concentrations (IC50s) for the most potent TOB analogues (C12 and C14) against A549 and Beas 2B cells were 4- to 64-fold and 32- to 64-fold higher, respectively, than their antifungal MIC values against various fungi. Unlike conventional aminoglycoside antibiotics, TOB analogues with alkyl chain lengths of C12 and C14 appear to inhibit fungi by inducing apoptosis and disrupting the fungal membrane as a novel mechanism of action. Amphiphilic TOB analogues showed broad-spectrum antifungal activities with minimal mammalian cell cytotoxicity. This study provides novel lead compounds for the development of antifungal drugs.

Tobramycin Variants with Enhanced Ribosome-Targeting Activity.

With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with 1) enhanced antimicrobial activity, 2) the ability to evade resistance mechanisms, and 3) the capability of targeting the ribosome with higher efficiency is more and more pressing. The chemical derivatization of the naturally occurring tobramycin (TOB) by attachment of 37 different thioether groups at the 6''-position led to the identification of generally poorer substrates of TOB-targeting AG-modifying enzymes (AMEs). Thirteen of these displayed better antibacterial activity than the parent TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity.

Effects of structural modifications on the metal binding, anti-amyloid activity, and cholinesterase inhibitory activity of chalcones.

As the number of individuals affected with Alzheimer's disease (AD) increases and the availability of drugs for AD treatment remains limited, the need to develop effective therapeutics for AD becomes more and more pressing. Strategies currently pursued include inhibiting acetylcholinesterase (AChE) and targeting amyloid-ß (Aß) peptides and metal-Aß complexes. This work presents the design, synthesis, and biochemical evaluation of a series of chalcones, and assesses the relationship between their structures and their ability to bind metal ions and/or Aß species, and inhibit AChE/BChE activity. Several chalcones were found to exhibit potent disaggregation of pre-formed N-biotinyl Aß1-42 (bioAß42) aggregates in vitro in the absence and presence of Cu(2+)/Zn(2+), while others were effective at inhibiting the action of AChE.

Discovery of inhibitors of Bacillus anthracis primase DnaG.

Primase DnaG is an essential bacterial enzyme that synthesizes short ribonucleotide primers required for chromosomal DNA replication. Inhibitors of DnaG can serve as leads for development of new antibacterials and biochemical probes. We recently developed a nonradioactive in vitro primase-pyrophosphatase assay to identify and analyze DnaG inhibitors. Application of this assay to DnaG from Bacillus anthracis (Ba DnaG), a dangerous pathogen, yielded several inhibitors, which include agents with DNA intercalating properties (doxorubicin and tilorone) as well as those that do not intercalate into DNA (suramin). A polyanionic agent and inhibitor of eukaryotic primases, suramin, identified by this assay as a low-micromolar Ba DnaG inhibitor, was recently shown to be also a low-micromolar inhibitor of Mycobacterium tuberculosis DnaG (Mtb DnaG). In contrast, another low-micromolar Ba DnaG inhibitor, tilorone, is much more potent against Ba DnaG than against Mtb DnaG, despite homology between these enzymes, suggesting that DnaG can be targeted selectively.