Inhibition of pathogenic bacterial carbonic anhydrases by monothiocarbamates.

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195-0.959 µM and of 0.149-1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.

In vivo efficacy of acetazolamide in a mouse model of Neisseria gonorrhoeae infection.

Gonococcal infections represent an urgent public health threat worldwide due to the increasing incidence of infections that has been accompanied by an increase in bacterial resistance to most antibiotics. This has resulted in a dwindling number of effective treatment options. Undoubtedly, there is a critical need to develop new, effective anti-gonococcal agents. In an effort to discover new anti-gonococcal therapeutics, we previously identified acetazolamide, a carbonic anhydrase inhibitor, as a novel inhibitor of Neisseria gonorrhoeae. Acetazolamide exhibited potent anti-gonococcal activity in vitro as it inhibited growth of strains of N. gonorrhoeae at concentrations that ranged from 0.5 to 4 µg/mL. The aim of this study was to investigate the in vivo efficacy of acetazolamide in a mouse model of N. gonorrhoeae genital tract infection. Compared to vehicle-treated mice, acetazolamide significantly reduced the gonococcal burden by 90% in the vagina of infected mice after three days of treatment. These results indicate that acetazolamide warrants further investigation as a promising treatment option to supplement the limited pipeline of anti-gonococcal therapeutics.

Dithiocarbamates effectively inhibit the α-carbonic anhydrase from Neisseria gonorrhoeae.

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the α-class carbonic anhydrase (CA, EC 4.2.1.1) from Neisseria gonorrhoeae, NgCA. As an extension to our previous studies, we report that dithiocarbamates (DTCs) derived from primary or secondary amines constitute a class of efficient inhibitors of NgCA. KIs ranging between 83.7 and 827 nM were measured for a series of 31 DTCs that incorporated various aliphatic, aromatic, and heterocyclic scaffolds. A subset of DTCs were selected for antimicrobial testing against N. gonorrhoeae, and three molecules displayed minimum inhibitory concentration (MIC) values less than or equal to 8 µg/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents.

Structure-activity relationship studies for inhibitors for vancomycin-resistant Enterococcus and human carbonic anhydrases.

Vancomycin-resistant enterococci (VRE), consisting of pathogenic Enterococcus faecalis and E. faecium, is a leading cause of hospital-acquired infections (HAIs). We recently repurposed the FDA-approved human carbonic anhydrase (CA) inhibitor acetazolamide (AZM) against VRE agent with the likely mechanism of action for the molecules being inhibition of one, or both, of the bacterial CA isoforms expressed in VRE. To elucidate how inhibitor binding to the enzymes relates to MIC, we further characterised the inhibition constants (Ki) against the E. faecium α-CA (Efα-CA) and γ-CA (Efγ-CA), as well as against human CA I (hCAI) and human CA II (hCAII) to assess selectivity. We have also utilised homology modelling and molecular dynamics (MD) simulations to gain a better understanding of the potential interactions the molecules are making with the targets. In this paper, we elaborate on the SAR for the AZM analogs as it pertains to MIC and Ki for each CA.

Repurposing FDA-approved sulphonamide carbonic anhydrase inhibitors for treatment of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.

Isoquinoline Antimicrobial Agent: Activity against Intracellular Bacteria and Effect on Global Bacterial Proteome.

A new class of alkynyl isoquinoline antibacterial compounds, synthesized via Sonogashira coupling, with strong bactericidal activity against a plethora of Gram-positive bacteria including methicillin- and vancomycin-resistant Staphylococcus aureus (S. aureus) strains is presented. HSN584 and HSN739, representative compounds in this class, reduce methicillin-resistant S. aureus (MRSA) load in macrophages, whilst vancomycin, a drug of choice for MRSA infections, was unable to clear intracellular MRSA. Additionally, both HSN584 and HSN739 exhibited a low propensity to develop resistance. We utilized comparative global proteomics and macromolecule biosynthesis assays to gain insight into the alkynyl isoquinoline mechanism of action. Our preliminary data show that HSN584 perturb S. aureus cell wall and nucleic acid biosynthesis. The alkynyl isoquinoline moiety is a new scaffold for the development of potent antibacterial agents against fatal multidrug-resistant Gram-positive bacteria.

In Vivo Antibacterial Activity of Acetazolamide.

Vancomycin-resistant enterococci (VRE) represent a major public health threat that requires the development of new therapeutics. In the present study, acetazolamide (AZM) was evaluated against enterococci. It inhibited different enterococcal strains tested at clinically achievable concentrations. Moreover, AZM outperformed linezolid, the drug of choice for VRE infections, in two in vivo VRE mouse models-murine colonization-reduction and VRE septicemia. Collectively, these results indicate that AZM warrants consideration as a promising treatment option for VRE infections.

N-(1,3,4-Oxadiazol-2-yl)Benzamides as Antibacterial Agents against Neisseria gonorrhoeae.

The Centers for Disease Control and Prevention (CDC) recognizes Neisseria gonorrhoeae as an urgent-threat Gram-negative bacterial pathogen. Additionally, resistance to frontline treatment (dual therapy with azithromycin and ceftriaxone) has led to the emergence of multidrug-resistant N. gonorrhoeae, which has caused a global health crisis. The drug pipeline for N. gonorrhoeae has been severely lacking as new antibacterial agents have not been approved by the FDA in the last twenty years. Thus, there is a need for new chemical entities active against drug-resistant N. gonorrhoeae. Trifluoromethylsulfonyl (SO2CF3), trifluoromethylthio (SCF3), and pentafluorosulfanyl (SF5) containing N-(1,3,4-oxadiazol-2-yl)benzamides are novel compounds with potent activities against Gram-positive bacterial pathogens. Here, we report the discovery of new N-(1,3,4-oxadiazol-2-yl)benzamides (HSGN-237 and -238) with highly potent activity against N. gonorrhoeae. Additionally, these new compounds were shown to have activity against clinically important Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Listeria monocytogenes (minimum inhibitory concentrations (MICs) as low as 0.25 µg/mL). Both compounds were highly tolerable to human cell lines. Moreover, HSGN-238 showed an outstanding ability to permeate across the gastrointestinal tract, indicating it would have a high systemic absorption if used as an anti-gonococcal therapeutic.

Repurposing the Antiamoebic Drug Diiodohydroxyquinoline for Treatment of Clostridioides difficile Infections.

Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at concentrations of 0.5 µg/ml and 2 µg/ml, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3 log10 within 6 h. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficilein vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit the growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium, and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

ß,γ-Diaryl α-methylene-γ-butyrolactones as potent antibacterials against methicillin-resistant Staphylococcus aureus.

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) synthesized via allylboration or allylindation reactions were screened against methicillin-resistant Staphylococcus aureus (MRSA) USA300. Unlike natural AMGBLs, such as parthenolide, synthetic analogs bearing aryl moieties at the ß- and γ-positions are potent against MRSA. The most potent molecules were comparable to vancomycin and linezolid, the drugs of the last resort for MRSA infections, in their effectiveness with minimum inhibitory concentrations (MICs) ranging from 3.0 to 5.2 µM. These lactones also exhibited potent antibacterial activity against other clinically important multidrug-resistant Gram-positive bacteria (except enterococci), while also showing high tolerability to mammalian cells. Several of these molecules surpassed vancomycin in their rapid killing of the high MRSA inoculum (2 h vs 12 h) in a standard time-kill kinetics assay, with compounds 1l and 1m significantly reducing the intracellular burden of MRSA by about 98-99%, at low concentrations. Additionally, the compounds surpassed vancomycin in inhibiting staphylococcal protease production, indicating that synthetic methylene lactones warrant further investigations as promising anti-MRSA candidates.

Synthesis and antimicrobial evaluation of new halogenated 1,3-Thiazolidin-4-ones.

The ongoing prevalence of multidrug-resistant bacterial pathogens requires the development of new effective antibacterial agents. In this study, two series of halogenated 1,3-thiazolidin-4-ones were synthesized and characterized. All the synthesized thiazolidinone derivatives were evaluated for their antimicrobial activity. Biological screening of the tested compounds revealed the antibacterial activity of the chlorinated thiazolidinones 4a, 4b and 4c against Escherichia coli TolC-mutant, with MIC values of 16 µg/mL. A combination of a sub-inhibitory concentration of colistin (0.25 × MIC) with compounds 4a, 4b or 4c showed antibacterial activity against different Gram-negative bacteria (MICs = 4-16 µg/mL). Interestingly, compounds 4a, 4b and 4c were not cytotoxic to murine fibroblasts and Caco-2 cells. The chlorinated thiazolidinone derivative 16d demonstrated a bacteriostatic activity against a panel of pathogenic Gram-positive bacteria, including clinical isolates of methicillin and vancomycin-resistant Staphylococcus aureus, Listeria monocytogenes and multidrug-resistant Staphylococcus epidermidis (MICs = 8 - 64 µg/mL), with no cytotoxicity against both Caco-2 and L929 cells. Compound 16d was superior to vancomycin in disruption of the pre-formed MRSA biofilm. Furthermore, the three fluorinated thiazolidinone derivatives 26c, 30c and 33c showed a hindrance to hemolysin activity, without cytotoxicity against L929 cells.

Antibiotic Susceptibility Determination within One Cell Cycle at Single-Bacterium Level by Stimulated Raman Metabolic Imaging.

The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. Therefore, there is an urgent need for a rapid method that can test the susceptibility of bacteria in a culture-free manner. Here we demonstrate a rapid AST method by monitoring the glucose metabolic activity of live bacteria at the single-cell level with hyperspectral stimulated Raman scattering (SRS) imaging. Using vancomycin-susceptible and -resistant enterococci E. faecalis as models, we demonstrate that the metabolic uptake of deuterated glucose in a single living bacterium can be quantitatively monitored via hyperspectral SRS imaging. Remarkably, the metabolic activity of susceptible bacteria responds differently to antibiotics from the resistant strain within only 0.5 h from the addition of antibiotics. Therefore, bacterial susceptibility and the minimum inhibitory concentration (MIC) of antibiotics can be determined within one cell cycle. Our metabolic imaging method is applicable to other bacteria species including E. coli, K. Pneumoniae, and S. aureus as well as different antibiotics, regardless of their mechanisms of inhibiting or killing bacteria.

Novel phenylthiazoles with a tert-butyl moiety: promising antimicrobial activity against multidrug-resistant pathogens with enhanced ADME properties.

The structure-activity relationship of a new tert-butylphenylthiazole series, with a pyrimidine linker, was investigated. We wished to expand knowledge of this novel class of antibiotics by generating 21 new derivatives bearing ≥2 heteroatoms in their side chains. Their activity was examined against isolates of methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Escherichia coli, Neisseria gonorrhoeae, and Candida albicans. Two compounds with 1,2-diaminocyclohexane as a nitrogenous side chain showed promising activity against the highly infectious MRSA USA300 strain, with a minimum inhibitory concentration (MIC) of 4 µg mL-1. One of these two compounds demonstrated potent activity against C. difficile, with a MIC of 4 µg mL-1. Moderate activities against a C. difficile strain with a MIC of 8 µg mL-1 were noted. Some new compounds possessed antifungal activity against a wild fluconazole-resistant C. albicans strain, with MIC values of 4-16 µg mL-1. ADME and metabolism-simulation studies were performed for the most promising compound and compared with lead compounds. Our results revealed that one compound possessed greater penetration of bacterial membranes and metabolic resistance, which aided a longer duration of action against MRSA.

Development of Penicillin-Based Carbonic Anhydrase Inhibitors Targeting Multidrug-Resistant Neisseria gonorrhoeae.

The development of antibacterial drugs with new mechanisms of action is crucial in combating the rise of antibiotic-resistant infections. Bacterial carbonic anhydrases (CAs, EC 4.2.1.1) have been validated as promising antibacterial targets against pathogens such as Helicobacter pylori, Neisseria gonorrhoeae, and vancomycin-resistant enterococci. A multitarget strategy is proposed to design penicillin-based CA inhibitor hybrids for tackling resistance by targeting multiple bacterial pathways, thereby resensitizing drug-resistant strains to clinical antibiotics. The sulfonamide derivatives potently inhibited the CAs from N. gonorrhoeae and Escherichia coli with KI values in the range of 7.1-617.2 nM. Computational simulations with the main penicillin-binding protein (PBP) of N. gonorrhoeae indicated that these hybrid derivatives maintained the mechanism of action of the lead ß-lactams. A subset of derivatives showed potent PBP-related antigonococcal effects against multidrug-resistant N. gonorrhoeae strains, with several compounds significantly outperforming both the lead ß-lactam and CA inhibitor drugs (MIC values in the range 0.25 to 0.5 µg/mL).

Colonization efficiency of multidrug-resistant Neisseria gonorrhoeae in a female mouse model.

The rapid occurrence of gonococcal resistance to all classes of antibiotics could lead to untreatable gonorrhea. Thus, development of novel anti-Neisseria gonorrhoeae drugs is urgently needed. Neisseria gonorrhoeae FA1090 is the most used in gonococcal infection mouse models because of its natural resistance to streptomycin. Streptomycin inhibits the urogenital commensal flora that permits gonococcal colonization. However, this strain is drug-susceptible and cannot be used to investigate the efficacy of novel agents against multidrug-resistant N. gonorrhoeae. Hence, to test the in vivo efficacy of new therapeutics against N. gonorrhoeae resistant to the frontline antibiotics, azithromycin, or ceftriaxone, we constructed streptomycin-resistant mutants of N. gonorrhoeae CDC-181 (azithromycin-resistant) and WHO-X (ceftriaxone-resistant). We identified the inoculum size needed to successfully colonize mice. Both mutants, CDC-181-rpsLA128G and WHO-X-rpsLA128G, colonized the genital tract of mice for 14 days with 100% colonization observed for at least 7 days. CDC-181-rpsLA128G demonstrated better colonization of the murine genital tract compared to WHO-X-rpsLA128G. Lower inoculum of WHO-X-rpsLA128G (105 and 106 CFU) colonized mice better than higher inoculum. Overall, our results indicate that CDC-181-rpsLA128G and WHO-X-rpsLA128G can colonize the lower genital tract of mice and are suitable to be used in mouse models to investigate the efficacy of antigonococcal agents.

Lopinavir and ritonavir act synergistically with azoles against Candida auris in vitro and in a mouse model of disseminated candidiasis.

INTRODUCTION AND OBJECTIVES: The emergence of Candida auris has created a global health challenge. Azole antifungals are the most affected antifungal class because of the extraordinary capability of C. auris to develop resistance against these drugs. Here, we used a combinatorial therapeutic approach to sensitize C. auris to azole antifungals. METHODS AND RESULTS: We have demonstrated the capability of the HIV protease inhibitors lopinavir and ritonavir, at clinically relevant concentrations, to be used with azole antifungals to treat C. auris infections both in vitro and in vivo. Both lopinavir and ritonavir exhibited potent synergistic interactions with the azole antifungals, particularly with itraconazole against 24/24 (100%) and 31/34 (91%) of tested C. auris isolates, respectively. Furthermore, ritonavir significantly interfered with the fungal efflux pump, resulting in a significant increase in Nile red fluorescence by 44%. In a mouse model of C. auris systemic infection, ritonavir boosted the activity of lopinavir to work synergistically with fluconazole and itraconazole and significantly reduced the kidney fungal burden by a 1.2 log (∼94%) and 1.6 log (∼97%) CFU, respectively. CONCLUSION: Our results urge further comprehensive assessment of azoles and HIV protease inhibitors as a novel drug regimen for the treatment of serious invasive C. auris infections.

Evaluation of 1,3,4-Thiadiazole Carbonic Anhydrase Inhibitors for Gut Decolonization of Vancomycin-Resistant Enterococci.

Vancomycin-resistant enterococci (VRE), Enterococcus faecium and Enterococcus faecalis, are high-priority drug-resistant pathogens in need of new therapeutic approaches. VRE originate in the gastrointestinal tract of carriers and can lead to more problematic downstream infections in the healthcare setting. Having a carrier of VRE admitted into a healthcare setting increases the risk to other patients for acquiring an infection. One strategy to eliminate the downstream infections is decolonization of VRE from carriers. Here, we report the activity of a set of carbonic anhydrase inhibitors in the in vivo VRE gastrointestinal decolonization mouse model. The molecules encompass a range of antimicrobial potency and intestinal permeability, and these factors were shown to influence the in vivo efficacy for VRE gut decolonization. Overall, carbonic anhydrase inhibitors exhibited superior VRE decolonization efficacy compared to the current drug of choice, linezolid.

Expanding the structure-activity relationships of alkynyl diphenylurea scaffold as promising antibacterial agents.

With the continuous and alarming threat of exhausting the current antimicrobial arsenals, efforts are urgently needed to develop new effective ones. In this study, the antibacterial efficacy of a set of structurally related acetylenic-diphenylurea derivatives carrying the aminoguanidine moiety was tested against a panel of multidrug-resistant Gram-positive clinical isolates. Compound 18 was identified with a superior bacteriological profile than the lead compound I. Compound 18 demonstrated an excellent antibacterial profile in vitro: low MIC values, extended post-antibiotic effect, refractory ability to resistance development upon extended repeated exposure, and high tolerability towards mammalian cells. Finally, when assessed in a MRSA skin infection animal model, compound 18 showed considerable healing and less inflammation, decrease in the bacterial loads in skin lesions, and it surpassed fusidic acid in controlling the systemic dissemination of S. aureus. Collectively, compound 18 represents a promising lead anti-MRSA agent that merits further investigation for the development of new anti-staphylococcal therapeutics.

Substituted salicylic acid analogs offer improved potency against multidrug-resistant Neisseria gonorrhoeae and good selectivity against commensal vaginal bacteria.

Drug-resistant Neisseria gonorrhoeae represents a major threat to public health; without new effective antibiotics, untreatable gonococcal infections loom as a real possibility. In a previous drug-repurposing study, we reported that salicylic acid had good potency against azithromycin-resistant N. gonorrhoeae. We now report that the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution, but that select substituted naphthyl analogs (3b, 3o and 3p) have superior activity to salicylic acid itself. Furthermore, these compounds retained potency against multiple ceftriaxone- and azithromycin-resistant strains, exhibited rapid bactericidal activity against N. gonorrhoeae, and showed high tolerability to mammalian cells (CC50 > 128 µg/mL). Promisingly, these compounds also show very weak growth inhibition of commensal vaginal bacteria.

Naphthylthiazoles: a class of broad-spectrum antifungals.

Cryptococcal infections remain a major cause of mortality worldwide due to the ability of Cryptococci to pass through the blood-brain barrier (BBB) causing lethal meningitis. The limited number of available therapeutics, which exhibit limited availability, severe toxicity and low tolerability, necessitates the development of new therapeutics. Investigating the antifungal activity of a novel series of naphthylthiazoles provided trans-diaminocyclohexyl derivative 18 with many advantageous attributes as a potential therapeutic for cryptococcal meningitis. Briefly, the antimycotic activity of 18 against cryptococcal strains was highly comparable to that of amphotericin-B and fluconazole with MIC values as low as 1 µg mL-1. Moreover, compound 18 possessed additional advantages over fluconazole; it significantly reduced the intracellular burden of Cryptococci and markedly inhibited cryptococcal biofilm formation. Initial PK assessment of 18 indicated its ability to reach the CNS after oral administration with high permeability, and it maintained therapeutic plasma concentrations for 18 h. Its antifungal activity extended to other clinically relevant strains, such as fluconazole-resistant C. auris.