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.

Discovery and development of inhibitors of acetyltransferase Eis to combat Mycobacterium tuberculosis.

Aminoglycosides are bactericidal antibiotics with a broad spectrum of activity, used to treat infections caused mostly by Gram-negative pathogens and as a second-line therapy against tuberculosis. A common resistance mechanism to aminoglycosides is bacterial aminoglycoside acetyltransferase enzymes (AACs), which render aminoglycosides inactive by acetylating their amino groups. In Mycobacterium tuberculosis, an AAC called Eis (enhanced intracellular survival) acetylates kanamycin and amikacin. When upregulated as a result of mutations, Eis causes clinically important aminoglycoside resistance; therefore, Eis inhibitors are attractive as potential aminoglycoside adjuvants for treatment of aminoglycoside-resistant tuberculosis. For over a decade, we have studied Eis and discovered several series of Eis inhibitors. Here, we provide a detailed protocol for a colorimetric assay used for high-throughput discovery of Eis inhibitors, their characterization, and testing their selectivity. We describe protocols for in vitro cell culture assays for testing aminoglycoside adjuvant properties of the inhibitors. A procedure for obtaining crystals of Eis-inhibitor complexes and determining their structures is also presented. Finally, we discuss applicability of these methods to discovery and testing of inhibitors of other AACs.

Small molecule inhibitors of the fosfomycin resistance enzyme FosM from Mycobacterium abscessus.

Fosfomycin is a safe broad-spectrum antibiotic that has not achieved widespread use because of the emergence of fosfomycin-modifying enzymes. Inhibition of fosfomycin-modifying enzymes could be used to help combat pathogens like Mycobacterium abscessus. Our previous work identified several inhibitors for the enzyme FosB from Staphylococcus aureus. We have tested those same compounds for inhibition of FosM, the fosfomycin-modifying enzyme from M. abscessus. The work described here will be used as the basis for more detailed studies into the inhibition of FosM.

Discovery and development of novel substituted monohydrazides as potent antifungal agents.

Novel substituted monohydrazides synthesized for this study displayed broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains. The activity of these compounds was either comparable or superior to amphotericin B against most of the fungal strains tested. These compounds possessed fungistatic activity in a time-kill assay and exhibited no mammalian cell toxicity. In addition, they prevented the formation of fungal biofilms. Even after repeated exposures, the Candida albicans ATCC 10231 (strain A) fungal strain did not develop resistance to these monohydrazides.

Identification and analysis of small molecule inhibitors of FosB from Staphylococcus aureus.

Antimicrobial resistance (AMR) poses a significant threat to human health around the world. Though bacterial pathogens can develop resistance through a variety of mechanisms, one of the most prevalent is the production of antibiotic-modifying enzymes like FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. FosB enzymes are found in pathogens such as Staphylococcus aureus, one of the leading pathogens in deaths associated with AMR. fosB gene knockout experiments establish FosB as an attractive drug target, showing that the minimum inhibitory concentration (MIC) of fosfomycin is greatly reduced upon removal of the enzyme. Herein, we have identified eight potential inhibitors of the FosB enzyme from S. aureus by applying high-throughput in silico screening of the ZINC15 database with structural similarity to phosphonoformate, a known FosB inhibitor. In addition, we have obtained crystal structures of FosB complexes to each compound. Furthermore, we have kinetically characterized the compounds with respect to inhibition of FosB. Finally, we have performed synergy assays to determine if any of the new compounds lower the MIC of fosfomycin in S. aureus. Our results will inform future studies on inhibitor design for the FosB enzymes.

Aromatic hydrazides: A potential solution for Acinetobacter baumannii infections.

The emergence of multidrug-resistant bacteria and the poor efficacy of available antibiotics against these infections have led to the urgent need for novel antibiotics. Acinetobacter baumannii is one of high-priority pathogens due to its ability to mount resistance to different classes of antibiotics. In an effort to provide novel agents in the fight against infections caused by A. baumannii, we synthesized a series of 46 aromatic hydrazides as potential treatments. In this series, 34 compounds were found to be low- to sub-µM inhibitors of A. baumannii growth, with MIC values in the range of 8 µg/mL to ≤0.125 µg/mL against a broad set of multidrug-resistant clinical isolates. These compounds were not highly active against other bacteria. We showed that one of the most potent compounds, 3e, was bacteriostatic and inhibitory to biofilm formation, although it did not disrupt the preformed biofilm. Additionally, we found that these compounds lacked mammalian cytotoxicity. The high antibacterial potency and the lack of mammalian cytotoxicity make these compounds a promising lead series for development of a novel selective anti-A. baumannii 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.

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 of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity.

A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 µM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

Discovery and Optimization of 6-(1-Substituted pyrrole-2-yl)-s-triazine Containing Compounds as Antibacterial Agents.

Antimicrobial drug resistance is a major health issue plaguing healthcare worldwide and leading to hundreds of thousands of deaths globally each year. Tackling this problem requires discovery and development of new antibacterial agents. In this study, we discovered novel 6-(1-substituted pyrrole-2-yl)-s-triazine containing compounds that potently inhibited the growth of Staphylococcus aureus regardless of its methicillin-resistant status, displaying minimum inhibitory concentration (MIC) values as low as 1 µM. The presence of a single imidazole substituent was critical to the antibacterial activity of these compounds. Some of the compounds also inhibited several nontubercular mycobacteria. We have shown that these molecules are potent bacteriostatic agents and that they are nontoxic to mammalian cells at relevant concentrations. Further development of these compounds as novel antimicrobial agents will be aimed at expanding our armamentarium of antibiotics.

Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut.

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

Structure-based design of haloperidol analogues as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis to overcome kanamycin resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.

Development of Single-Stranded DNA Bisintercalating Inhibitors of Primase DnaG as Antibiotics.

Many essential enzymes in bacteria remain promising potential targets of antibacterial agents. In this study, we discovered that dequalinium, a topical antibacterial agent, is an inhibitor of Staphylococcus aureus primase DnaG (SaDnaG) with low-micromolar minimum inhibitory concentrations against several S. aureus strains, including methicillin-resistant bacteria. Mechanistic studies of dequalinium and a series of nine of its synthesized analogues revealed that these compounds are single-stranded DNA bisintercalators that penetrate a bacterium by compromising its membrane. The best compound of this series likely interacts with DnaG directly, inhibits both staphylococcal cell growth and biofilm formation, and displays no significant hemolytic activity or toxicity to mammalian cells. This compound is an excellent lead for further development of a novel anti-staphylococcal therapeutic.

Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from Mycobacterium tuberculosis.

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.

Probing the Robustness of Inhibitors of Tuberculosis Aminoglycoside Resistance Enzyme Eis by Mutagenesis.

Each year, millions of people worldwide contract tuberculosis (TB), the deadliest infection. The spread of infections with drug-resistant strains of Mycobacterium tuberculosis (Mtb) that are refractory to treatment poses a major global challenge. A major cause of resistance to antitubercular drugs of last resort, aminoglycosides, is overexpression of the Eis (enhanced intracellular survival) enzyme of Mtb, which inactivates aminoglycosides by acetylating them. We showed previously that this inactivation of aminoglycosides could be overcome by our recently reported Eis inhibitors that are currently in development as potential aminoglycoside adjunctive therapeutics against drug-resistant TB. To interrogate the robustness of the Eis inhibitors, we investigated the enzymatic activity of Eis and its inhibition by Eis inhibitors from three different structural families for nine single-residue mutants of Eis, including those found in the clinic. Three engineered mutations of the substrate binding site, D26A, W36A, and F84A, abolished inhibitor binding while compromising Eis enzymatic activity 2- to 3-fold. All other Eis mutants, including clinically observed ones, were potently inhibited by at least one inhibitor. This study helps position us one step ahead of Mtb resistance to Eis inhibitors as they are being developed for TB therapy.

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.

N,N'-diaryl-bishydrazones in a biphenyl platform: Broad spectrum antifungal agents.

N,N'-Diaryl-bishydrazones of [1,1'-biphenyl]-3,4'-dicarboxaldehyde, [1,1'-biphenyl]-4,4'-dicarboxaldehyde, and 4,4'-bisacetyl-1,1-biphenyl exhibited excellent antifungal activity against a broad spectrum of filamentous and non-filamentous fungi. These N,N'-diaryl-bishydrazones displayed no antibacterial activity in contrast to previously reported N,N'-diamidino-bishydrazones and N-amidino-N'-aryl-bishydrazones. The leading candidate, 4,4'-bis((E)-1-(2-(4-fluorophenyl)hydrazono)ethyl)-1,1'-biphenyl, displayed less hemolysis of murine red blood cells at concentrations at or below that of a control antifungal agent (voriconazole), was fungistatic in a time-kill study, and possessed no mammalian cytotoxicity and no toxicity with respect to hERG inhibition.

Synthesis, antimicrobial activity, attenuation of aminoglycoside resistance in MRSA, and ribosomal A-site binding of pyrene-neomycin conjugates.

The development of new ligands that have comparable or enhanced therapeutic efficacy relative to current drugs is vital to the health of the global community in the short and long term. One strategy to accomplish this goal is to functionalize sites on current antimicrobials to enhance specificity and affinity while abating resistance mechanisms of infectious organisms. Herein, we report the synthesis of a series of pyrene-neomycin B (PYR-NEO) conjugates, their binding affinity to A-site RNA targets, resistance to aminoglycoside-modifying enzymes (AMEs), and antibacterial activity against a wide variety of bacterial strains of clinical relevance. PYR-NEO conjugation significantly alters the affinities of NEO for bacterial A-site targets. The conjugation of PYR to NEO significantly increased the resistance of NEO to AME modification. PYR-NEO conjugates exhibited broad-spectrum activity towards Gram-positive bacteria, including improved activity against NEO-resistant methicillin-resistant Staphylococcus aureus (MRSA) strains.

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.

Interfering With DNA Decondensation as a Strategy Against Mycobacteria.

Tuberculosis is once again a major global threat, leading to more than 1 million deaths each year. Treatment options for tuberculosis patients are limited, expensive and characterized by severe side effects, especially in the case of multidrug-resistant forms. Uncovering novel vulnerabilities of the pathogen is crucial to generate new therapeutic strategies. Using high resolution microscopy techniques, we discovered one such vulnerability of Mycobacterium tuberculosis. We demonstrate that the DNA of M. tuberculosis can condense under stressful conditions such as starvation and antibiotic treatment. The DNA condensation is reversible and specific for viable bacteria. Based on these observations, we hypothesized that blocking the recovery from the condensed state could weaken the bacteria. We showed that after inducing DNA condensation, and subsequent blocking of acetylation of DNA binding proteins, the DNA localization in the bacteria is altered. Importantly under these conditions, Mycobacterium smegmatis did not replicate and its survival was significantly reduced. Our work demonstrates that agents that block recovery from the condensed state of the nucleoid can be exploited as antibiotic. The combination of fusidic acid and inhibition of acetylation of DNA binding proteins, via the Eis enzyme, potentiate the efficacy of fusidic acid by 10 and the Eis inhibitor to 1,000-fold. Hence, we propose that successive treatment with antibiotics and drugs interfering with recovery from DNA condensation constitutes a novel approach for treatment of tuberculosis and related bacterial infections.