Mechanism-based inhibition of gut microbial tryptophanases reduces serum indoxyl sulfate.

Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.

Genetic Determinants of Acinetobacter baumannii Serum-Associated Adaptive Efflux-Mediated Antibiotic Resistance.

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism's adaptive efflux-mediated resistance phenotype.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

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.

Piperazinobenzodiazepinones: New Encephalitic Alphavirus Inhibitors via Ring Expansion of 2-Dichloromethylquinazolinones.

Venezuelan and eastern equine encephalitis viruses are disease-causing, neuropathic pathogens with no approved treatment options in humans. While expanding the pharmacophoric model of antialphaviral amidines prepared via a quinazolinone rearrangement, we discovered that diamine-treated, 2-dihalomethylquinolinones unexpectedly afforded ring-expanded piperazine-fused benzodiazepinones. Notably, this new chemotype (19 examples) showed potent, submicromolar inhibition of virus-induced cell death, >7-log reduction of viral yield, and tractable structure-activity relationships across both viruses. Antiviral activity was confirmed in primary human neuronal cells. A mechanistic rationale for product formation is proposed, and key structural elements were comparatively modeled between a similarly substituted antiviral amidine and piperazinobenzodiazepinone prototypes to guide future antiviral development.

3D Printing Ultraflexible Magnetic Actuators via Screw Extrusion Method.

Soft magnetic actuators with programmable structure design and controllable deformation ability based on 3D printing technology have attracted extensive attention. In this paper, a novel 3D printing strategy is developed to manufacture the ultraflexible magnetic actuator, in which the printed material is composed of magnetic particles and thermoplastic rubber materials. Different from the traditional fused deposition printing, this printing strategy introduces screw extrusion technology to the heating components of the printer to overcome the problem of filament buckling in the flexible material. Thus, the tensile modulus of the printed products can be reduced to as low as ≈2 MPa. Based on the above method, biomimetic magnetic actuators of the sucker and the pump are constructed for adhering and releasing object and pumping liquid. The contraction performance of the magnetic actuator is studied via a series of experiments and the magnetic field-induced deformation is analyzed by the multiphysics-based finite element model. This work proves that ultraflexible magnetic actuators fabricated by this 3D printing strategy show broad prospects in the fields of soft robotics and bionics.

3D Printing Magnetic Actuators for Biomimetic Applications.

Biomimetic actuators with stimuli-responsiveness, adaptivity, and designability have attracted extensive attention. Recently, soft intelligent actuators based on stimuli-responsive materials have been gradually developed, but it is still challenging to achieve various shape manipulations of actuators through a simple 3D printing technology. In this paper, a 3D printing strategy based on magneto-active materials is developed to manufacture various biomimetic magnetic actuators, in which the new printable magnetic filament is composed of a thermoplastic rubber material and magnetic particles. The continuous shape transformation of magnetic actuators is further demonstrated to imitate the motion characteristic of creatures, including the predation behavior of octopus tentacles, the flying behavior of the butterfly, and the flower blooming behavior of the plant. Furthermore, the magnetic field-induced deformation of the biomimetic structure can be simulated by the finite element method, which can further guide the structural design of the actuators. This work proves that the biomimetic actuator based on soft magneto-active materials has the advantages of programmable integrated structure, rapid prototyping, remote noncontact actuation, and rapid magnetic response. As a result, this 3D printing method possesses broad application prospects in soft robotics and other fields.

Optimization of 2-Acylaminocycloalkylthiophene Derivatives for Activity against Staphylococcus aureus RnpA.

Staphylococcus aureus is well-recognized to cause debilitating bacterial infections that are difficult to treat due to the emergence of antibiotic resistance. As such, there is a need to develop new antimicrobials for the therapeutic intervention of S. aureus disease. To that end, S. aureus RnpA is an essential enzyme that is hypothesized to participate in two required cellular processes, precursor tRNA (ptRNA) maturation and mRNA degradation. Corresponding high throughput screening campaigns have identified the phenylcarbamoyl cyclic thiopenes as a chemical class of RnpA inhibitors that display promising antibacterial effects by reducing RnpA ptRNA and mRNA degradation activities and low human cell toxicity. Herein, we perform a structure activity relationship study of the chemical scaffold. Results revealed that the cycloalkane ring size and trifluoroacetamide moiety are required for antibacterial activity, whereas modifications of the para and/or meta positions of the pharmacophore's phenyl group allowed tuning of the scaffold's antimicrobial performance and RnpA inhibitory activity. The top performing compounds with respect to antimicrobial activity also did not exhibit cytotoxicity to human cell lines at concentrations up to 100 µM, greater than 100-fold the minimum inhibitory concentration (MIC). Focused studies of one analog, RNP0012, which exhibited the most potent antimicrobial and inhibition of cellular RnpA activities revealed that the compound reduced bacterial burden in a murine model of S. aureus disease. Taken together, the results presented are expected to provide an early framework for optimization of next-generation of RnpA inhibitor analogues that may represent progenitors of a new class of antimicrobials.

Structure-Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria's essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 µg/mL equating to an 8- to 16-fold improvement in antigonococcal activity compared to acetazolamide. These analogues were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 µg/mL. Taken together, this study presents a class of antigonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.

Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 µg/mL (acetazolamide) to MIC = 0.007 µg/mL (22) and 1 µg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Optimization of 4-Substituted Benzenesulfonamide Scaffold To Reverse Acinetobacter baumannii Serum-Adaptive Efflux Associated Antibiotic Tolerance.

Acinetobacter baumannii is a nosocomial pathogen of urgent concern for public health due to rising rates of multidrug and pandrug resistance. In the context of environmental cues such as growth in human serum, A. baumannii is known to display adaptive efflux, in which a multitude of efflux-associated genes are upregulated, resulting in efflux-mediated drug tolerance in strains that are otherwise susceptible to antibiotic therapy. Previously, we identified a sulfonamide-containing scaffold molecule (ABEPI1) that reversed serum-associated antibiotic tolerance in A. baumannii. Herein, we present structure-activity relationship studies on 29 newly synthesized analogues. These molecules were characterized for their ability to potentiate multiple antibiotics in serum, reduce serum-associated ethidium bromide efflux and depolarize bacterial cell membranes. In addition, they were assessed for toxicity to mammalian cells. Collectively, these molecules may represent promising potential adjuvants for use in combination with new and existing antibiotics to treat A. baumannii bacterial infections.

Efficacy of a ML336 derivative against Venezuelan and eastern equine encephalitis viruses.

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.

Telescoped synthesis of C3-functionalized (E)-arylamidines using Ugi-Mumm and regiospecific quinazolinone rearrangements.

An efficient four-step, six-transformation protocol was developed to afford bioactive N-alkyl- or N-arylamide (E)-arylamidines featuring strategic amidine C3 modifications which were inaccessible or low yielding by previous methods. This synthetic approach, exemplified with 24 amidines and requiring only a single purification, highlights a multicomponent Ugi-Mumm rearrangement to afford highly diversified quinazolinones which undergo regiospecific rearrangement to afford new amidines. The method extensively broadens the structural scope of this new class of trisubstituted amidines and demonstrates the tolerance of regional C3 amidine steric bulk, visualized with X-ray crystallographic analysis.

Design, synthesis, and structure-activity relationship studies of novel tetrazole antifungal agents with potent activity, broad antifungal spectrum and high selectivity.

In this letter, we report our efforts to design, synthesize and evaluate biological activities of a series of novel hybridized compounds containing 1-tetrazole and 4-pyridinyl-1,2,4-triazole-3-one. An analysis of structure-activity data indicates that the target compounds with bulky and hydrophobic side chains exhibited stronger activities against the Candida spp and Cryptococcus neoformans tested than those of fluconazole and racemic VT-1161. Furthermore, 13k and 13ad were active against Microsporum gypseum, which was resistant to racemic VT-1161. In addition, 13k, 13ac and 13ad, with good in vitro activities against all of pathogenic fungi tested except for Aspergillus fumigatus, had no inhibition of human CYP3A4, suggesting a low risk of drug-drug interactions.

Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates.

In order to further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compounds (I), two series of novel azoles featuring thieno[2,3-c]pyrrolidone and thieno[3,2-c]pyrrolidone nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SARs of antifungal triazoles. Most of target compounds exhibited excellent activity against Candida and Cryptococcus spp., with MIC values in the range of 0.0625 µg/ml to 0.0156 µg/ml. The thieno[3,2-c]pyrrolidone unit was more suited for improving activity against Aspergillus spp. The most potent compound, 18a, was selected for further development due to its significant in vitro activity against Aspergillus spp. (MIC = 0.25 µg/ml), as well as its high metabolic stability in human liver microsomes.

Design, synthesis, and structure-activity relationship studies of novel fused heterocycles-linked triazoles with good activity and water solubility.

Triazoles with fused-heterocycle nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SAR of antifungal triazoles. Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine and tetrahydro-thiazolo[5,4-c]pyridine nuclei were preferable to the other four fused-heterocycle nuclei investigated. Potent in vitro activity, broad spectrum and better water solubility were attained when triazoles containing nitrogen aromatic heterocycles were attached to these two nuclei. The most potent compounds 27aa and 45x, with low hERG inhibition and hepatocyte toxicity, both exhibited excellent activity against Candida, Cryptococcus, and Aspergillus spp., as well as selected fluconazole-resistant strains. A high water-soluble compound 58 (the disulfate salt of 45x) displayed unsatisfactory in vivo activity because of its poor PK profiles. Mice infected with C.alb. SC5314 and C.alb. 103 (fluconazole-resistant strain) and administered with 27aa displayed significantly improved survival rates. 27aa also showed favorable pharmacokinetic (PK) profiles.

Design and synthesis of pyridine-substituted itraconazole analogues with improved antifungal activities, water solubility and bioavailability.

To improve antifungal activities, water solubility and bioavailability, a series of novel analogues of itraconazole-containing pyridine rings were designed and synthesized. Their antifungal activities were evaluated in vitro against six clinically important fungi by measuring the minimal inhibitory concentrations (MICs). Most of the compounds showed more potent antifungal activities than that of itraconazole. In particular, the analogues 30d, 30c, 31c, and 36d exhibited much higher solubility and bioavailability than that of itraconazole. The bioavailability of 36d (42.2%) was five times higher than that of itraconazole (8%) and was negative for genetic toxicology in the Ames test.