Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery.

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.

Modified HT-SPOTi: An Antimicrobial Susceptibility Testing to Evaluate Formulated Therapeutic Combinations Against Bacterial Growth and Viability.

Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.

Correction: Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0060143.].

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Antimicrobial Peptides Designed against the Ω-Loop of Class A ß-Lactamases to Potentiate the Efficacy of ß-Lactam Antibiotics.

Class A serine ß-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of ß-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of ß-lactamases and reduce their efficacy, along with potentiating the ß-lactam antibiotics and eventually decreasing ß-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the ß-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced ß-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and ß-lactams sensitised the bacteria towards the ß-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the ß-lactamases.

C2-linked alkynyl poly-ethylene glycol(PEG) adenosine conjugates as water-soluble adenosine receptor agonists.

A series of 12 novel polyethylene-glycol(PEG)-alkynyl C2-adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN-PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1 , A2A , A2B and A3 adenosine receptors (ARs) using crystal-structures and homology models. ADN-PEG-conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1 , A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN-PEG conjugates.

Synthesis, biological evaluation and computational studies of pyrazole derivatives as Mycobacterium tuberculosis CYP121A1 inhibitors.

A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-step synthetic scheme and evaluated for binding affinity with Mycobacterium tuberculosis (Mtb) CYP121A1 and antimycobacterial activity against Mtb H37Rv. Antimycobacterial susceptibility was measured using the spot-culture growth inhibition assay (SPOTi): the imidazoles displayed minimum inhibitory concentration (MIC90) in the range of 3.95-12.03 µg mL-1 (10.07-33.19 µM) with 11f the most active, while the triazoles displayed MIC90 in the range of 4.35-25.63 µg mL-1 (11.88-70.53 µM) with 12b the most active. Assessment of binding affinity using UV-vis spectroscopy showed that for the imidazole series, the propyloxy (11f) and isopropyloxy (11h) derivatives of the 4-chloroaryl pyrazoles displayed Mtb CYP121A1 type II binding affinity with K d 11.73 and 17.72 µM respectively compared with the natural substrate cYY (K d 12.28 µM), while in the triazole series, only the methoxy substitution with the 4-chloroaryl pyrazole (12b) showed good type II Mtb CYP121A1 binding affinity (K d 5.13 µM). Protein-detected 1D 19F-NMR spectroscopy as an orthogonal strategy was used to evaluate ligand binding independent of perturbations at the haem. For imidazole and triazole compounds, perturbations were more intense than cYY indicating tighter binding and confirming that ligand coordination occurs in the substrate-binding pocket despite very modest changes in UV-vis absorbance, consistent with computational studies and the demonstrated potential anti-tuberculosis properties of these compounds.

The Phytochemistry and Pharmacology of Tulbaghia, Allium, Crinum and Cyrtanthus: 'Talented' Taxa from the Amaryllidaceae.

Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer's disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates. Given the reciprocal relationship of the increasing burden of human diseases and limited availability of medicinal therapies, more rapid drug discovery and development are desirable. To expedite clinically relevant drug development, we present here evidence on bioactive compounds from the genera Allium, Tulgbaghia, Cyrtanthus and Crinum and describe their traditional and pharmacological applications.

Immunobiology of tubercle bacilli and prospects of immunomodulatory drugs to tackle tuberculosis (TB) and other non-tubercular mycobacterial infections.

The COVID-19 pandemic has set back progress made on antimicrobial resistance (AMR). Without urgent re-focus, we risk slowing down drug discovery and providing treatment for drug resistant Mycobacterium tuberculosis. Unique in its immune evasion, dormancy and resuscitation, the causal pathogens of tuberculosis (TB) have demonstrated resistance to antibiotics with efflux pumps and the ability to form biofilms. Repurposing drugs is a prospective avenue for finding new anti-TB drugs. There are many advantages to discovering novel targets of an existing drug, as the pharmacokinetic and pharmacodynamic properties have already been established, they are cost-efficient and can be commercially accelerated for the new development. One such group of drugs are non-steroidal anti-inflammatory drugs (NSAIDs) that are originally known for their ability to supress the host proinflammatory responses. In addition to their anti-inflammatory properties, some NSAIDs have been discovered to have antimicrobial modes of action. Of particular interest is Carprofen, identified to inhibit the efflux mechanism and disrupt biofilm formation in mycobacteria. Due to the complexities of host-pathogens interactions in the lung microbiome, inflammatory responses must carefully be controlled alongside the in vivo actions of the prospective anti-infectives. This critical review explores the potential dual role of a selection of NSAIDs, as an anti-inflammatory and anti-tubercular adjunct to reverse the tide of antimicrobial resistance in existing treatments.

Role of AmpC-Inducing Genes in Modulating Other Serine Beta-Lactamases in Escherichia coli.

The consistently mutating bacterial genotypes appear to have accelerated the global challenge with antimicrobial resistance (AMR); it is therefore timely to investigate certain less-explored fields of targeting AMR mechanisms in bacterial pathogens. One of such areas is beta-lactamase (BLA) induction that can provide us with a collection of prospective therapeutic targets. The key genes (ampD, ampE and ampG) to which the AmpC induction mechanism is linked are also involved in regulating the production of fragmented muropeptides generated during cell-wall peptidoglycan recycling. Although the involvement of these genes in inducing class C BLAs is apparent, their effect on serine beta-lactamase (serine-BLA) induction is little known. Here, by using ∆ampD and ∆ampE mutants of E. coli, we attempted to elucidate the effects of ampD and ampE on the expression of serine-BLAs originating from Enterobacteriaceae, viz., CTX-M-15, TEM-1 and OXA-2. Results show that cefotaxime is the preferred inducer for CTX-M-15 and amoxicillin for TEM-1, whereas oxacillin for OXA-2. Surprisingly, exogenous BLA expressions are elevated in ∆ampD and ∆ampE mutants but do not always alter their beta-lactam susceptibility. Moreover, the beta-lactam resistance is increased upon in trans expression of ampD, whereas the same is decreased upon ampE expression, indicating a differential effect of ampD and ampE overexpression. In a nutshell, depending on the BLA, AmpD amidase moderately facilitates a varying level of serine-BLA expression whereas AmpE transporter acts likely as a negative regulator of serine-BLA.