Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets.

Chemical genetics reveals a kinase-independent role for protein kinase R in pyroptosis.

Formation of the inflammasome, a scaffolding complex that activates caspase-1, is important in numerous diseases. Pyroptotic cell death induced by anthrax lethal toxin (LT) is a model for inflammasome-mediated caspase-1 activation. We discovered 7-desacetoxy-6,7-dehydrogedunin (7DG) in a phenotypic screen as a small molecule that protects macrophages from LT-induced death. Using chemical proteomics, we identified protein kinase R (PKR) as the target of 7DG and show that RNAi knockdown of PKR phenocopies treatment with 7DG. Further, we show that PKR's role in ASC assembly and caspase-1 activation induced by several different inflammasome stimuli is independent of PKR's kinase activity, demonstrating that PKR has a previously uncharacterized role in caspase-1 activation and pyroptosis that is distinct from its reported kinase-dependent roles in apoptosis and inflammasome formation in lipopolysaccharide-primed cells. Remarkably, PKR has different roles in two distinct cell death pathways and has a broad role in inflammasome function relevant in other diseases.

"Multiplexed screen identifies a Pseudomonas aeruginosa -specific small molecule targeting the outer membrane protein OprH and its interaction with LPS".

The surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa , a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT 1 , a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM ß-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal novel pathogen biology.

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

Synthesis and structure-activity relationships of phenyl-substituted coumarins with anti-tubercular activity that target FadD32.

In an effort to develop new and potent agents for therapy against tuberculosis, a high-throughput screen was performed against Mycobacterium tuberculosis strain H37Rv. Two 6-aryl-5,7-dimethyl-4-phenylcoumarin compounds 1a and 1b were found with modest activity. A series of coumarin derivatives were synthesized to improve potency and to investigate the structure-activity relationship of the series. Among them, compounds 1o and 2d showed improved activity with IC90 of 2 µM and 0.5 µM, respectively. Further optimization provided compound 3b with better physiochemical properties with IC90 0.4 µM which had activity in a mouse model of infection. The role of the conformation of the 4- and 6-aryl substituents is also described.

STAT6 phosphorylation inhibitors block eotaxin-3 secretion in bronchial epithelial cells.

The STAT6 (signal transducer and activator of transcription 6) protein facilitates T-helper cell 2 (Th2) mediated responses that control IgE-mediated atopic diseases such as asthma. We have identified compounds that bind to STAT6 and inhibit STAT6 tyrosine phosphorylation induced by IL-4. In the bronchial epithelial cell line BEAS-2B, compound (R)-84 inhibits the secretion of eotaxin-3, a chemokine eliciting eosinophil infiltration. (R)-84 appears to prevent STAT6 from assuming the active dimer configuration by directly binding the protein and inhibiting tyrosine phosphorylation.

Large-Scale Chemical-Genetic Strategy Enables the Design of Antimicrobial Combination Chemotherapy in Mycobacteria.

The efficacies of all antibiotics against tuberculosis are eventually eroded by resistance. New strategies to discover drugs or drug combinations with higher barriers to resistance are needed. Previously, we reported the application of a large-scale chemical-genetic interaction screening strategy called PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) for the discovery of new Mycobacterium tuberculosis inhibitors, which resulted in the identification of the small molecule BRD-8000, an inhibitor of a novel target, EfpA [ Johnson et al. ( 2019 ) Nature 517 , 72 ]. Leveraging the chemical genetic interaction profile of BRD-8000, we identified BRD-9327, another structurally distinct small molecule EfpA inhibitor. We show that the two compounds are synergistic and display collateral sensitivity because of their distinct modes of action and resistance mechanisms. High-level resistance to one increases the sensitivity to and reduces the emergence of resistance to the other. Thus, the combination of BRD-9327 and BRD-8000 represents a proof-of-concept for the novel strategy of leveraging chemical genetics in the design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is exploited.

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5'-azido-2',5'-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5'-amino-5'-deoxythymidine to the corresponding 5'-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension-cleavage procedure in genomic sequence analysis is discussed.

Baeyer-Villiger Monooxygenases EthA and MymA Are Required for Activation of Replicating and Non-replicating Mycobacterium tuberculosis Inhibitors.

Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least 6 months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Furthermore, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide-resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis.

Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation.

Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]

Asymmetric Addition of Alkyllithium to Chiral Imines: alpha-Naphthylethyl Group as a Chiral Auxiliary.

The diastereoselective nucleophilic addition of alkyllithium to N-alkylidene-alpha-naphthylethylamine was carried out. In the presence of Lewis acids or Lewis bases, organolithiums reacted smoothly with imines to give the corresponding amines in high stereoselectivity (up to 100% de). Furthermore, the resulting optically active amines were found to be useful for asymmetric reactions as chiral ligands.

Chiral Lewis Acid-Mediated Enantioselective Pictet-Spengler Reaction of N(b)-Hydroxytryptamine with Aldehydes.

The first example of a reagent-controlled enantioselective Pictet-Spengler reaction is demonstrated. Using diisopinocampheylchloroborane as a chiral Lewis acid catalyst, the Pictet-Spengler reaction of N(b)-hydroxytryptamine with aldehydes gave the corresponding 2-hydroxytetrahydro-beta-carbolines in up to 90% ee. The enantioselective Pictet-Spengler reaction catalyzed by chiral binaphthol-derived Brønsted acid-assisted Lewis acids, with up to 91% ee, is also demonstrated.

Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model.

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a nonreplicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the nonreplicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel nonreplicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and nonreplicating M. tuberculosis as well as compounds that inhibit the transition from nonreplicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of nonreplicating M. tuberculosis In order to understand the relationship among different nonreplicating models, we tested 52 of these molecules in a hypoxia model, and four different chemical scaffolds in a stochastic persister model, and a streptomycin-dependent model. We found that compounds display varying levels of activity in different models for the nonreplicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different nonreplicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development.

Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening.

Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

This unit presents synthetic procedures for the preparation of 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, and thymidine, as well as corresponding 5'-N-triphosphate nucleotides, using commercially available reagents. The modified nucleosides are prepared in high yields from naturally occurring 2'-deoxynucleosides using robust chemical reactions including tosylation, azide exchange, and the Staudinger reaction. Efficient conversion of these 5'-amino nucleosides to corresponding 5'-N-triphosphate nucleotides is achieved through a one-step reaction with trimetaphosphate in Tris-buffered aqueous solution. The 5'-amino modification renders these nucleoside and nucleotide analogs markedly increased reactivity, which is useful for a variety of biochemical, pharmaceutical, and genomic applications. Also included in this unit are protocols for polymerase incorporation of the 5'-amino nucleotides, either partially or completely replacing their naturally occurring counterparts, and subsequent sequence-specific cleavage at the modified nucleotides under mildly acidic conditions.

Sequence-specific dinucleotide cleavage promoted by synergistic interactions between neighboring modified nucleotides in DNA.

Sequence-specific cleavage of DNA by restriction endonucleases has been an indispensable tool in modern molecular biology. However, many potential applications are yet to be realized because of the limited number of naturally available restriction specificities. Efforts to expand this repertoire through protein engineering have met considerable challenges and only brought forth modest success. Taking an alternative approach, we developed a methodology to generate modified DNA susceptible to specific cleavage at selected dinucleotide sequences. This method requires the incorporation of two deoxyribonucleotide analogues by a DNA polymerase: a ribonucleotide and a 5'-amino-2',5'-dideoxyribonucleotide, each of which contains a different base. When linked in a 5' to 3' geometry, the two modified nucleotides act synergistically to promote cleavage at the phosphoramidate linkage, thus providing sequence specificity. Using the transferrin receptor gene as an example, we demonstrate that this dinucleotide cleavage generates discrete DNA fragments that can be either visualized by gel electrophoresis or detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides.

Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitro-dGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and/or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.