Scaffold Morphing To Identify Novel DprE1 Inhibitors with Antimycobacterial Activity.

We report a novel benzimidazole (BI) based DprE1 inhibitor that resulted from scaffold morphing of a 1,4-azaindole series. The clinical progression of the 1,4-azaindole series from our previous work validates the potential of exploring newer chemical entities with antimycobacterial activity driven via a noncovalent inhibition of the decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). The representative compounds from the new scaffold reported in this study exhibited an improved solubility and higher free plasma fraction, while retaining potent DprE1 inhibition and antimycobacterial activity. A representative compound from the benzimidazole series demonstrated good efficacy in a murine model of tuberculosis. Furthermore, molecular modeling of the BI scaffold suggests plausible modes of binding in the active site of DprE1 enzyme from Mycobacterium tuberculosis that can be used for further exploration of the series.

Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.

2-Phenylindole and Arylsulphonamide: Novel Scaffolds Bactericidal against Mycobacterium tuberculosis.

A cellular activity-based screen on Mycobacterium tuberculosis (Mtb) H37Rv using a focused library from the AstraZeneca corporate collection led to the identification of 2-phenylindoles and arylsulphonamides, novel antimycobacterial scaffolds. Both the series were bactericidal in vitro and in an intracellular macrophage infection model, active against drug sensitive and drug resistant Mtb clinical isolates, and specific to mycobacteria. The scaffolds showed promising structure-activity relationships; compounds with submicromolar cellular potency were identified during the hit to lead exploration. Furthermore, compounds from both scaffolds were tested for inhibition of known target enzymes or pathways of antimycobacterial drugs including InhA, RNA polymerase, DprE1, topoisomerases, protein synthesis, and oxidative-phosphorylation. Compounds did not inhibit any of the targets suggesting the potential of a possible novel mode of action(s). Hence, both scaffolds provide the opportunity to be developed further as leads and tool compounds to uncover novel mechanisms for tuberculosis drug discovery.

UDP-N-acetylmuramic acid l-alanine ligase (MurC) inhibition in a tolC mutant Escherichia coli strain leads to cell death.

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

1,4-azaindole, a potential drug candidate for treatment of tuberculosis.

New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.

Diarylthiazole: an antimycobacterial scaffold potentially targeting PrrB-PrrA two-component system.

Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE)>0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.

Lead optimization of 1,4-azaindoles as antimycobacterial agents.

In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.

4-aminoquinolone piperidine amides: noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Azaindoles: noncovalent DprE1 inhibitors from scaffold morphing efforts, kill Mycobacterium tuberculosis and are efficacious in vivo.

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: linking compound levels to cellular potency.

Dihydrofolate reductase (DHFR) plays a central role in maintaining cellular pool of tetrahydrofolic acid, a cofactor necessary for DNA, RNA and protein synthesis. The clinical validation of DHFR as antibacterial target was established by the success of trimethoprim (TMP). DHFR is also an attractive target for identifying anti-tuberculosis molecules however, due to observed weak cellular potency, no DHFR inhibitors have been developed as drugs so far. TMP and its analogs have poor cellular potency on Mycobacterium tuberculosis and Mycobacterium smegmatis cells. We found a mutant strain of M. smegmatis, mc²155 to be sensitive to TMP whereas wild type strain was not inhibited by TMP. We utilized this system to probe if poor or lack of activity of TMP is a consequence of poor intracellular compound levels. An LC-MS based method was developed for measuring TMP and rifampicin (RIF) in M. smegmatis. Using the assay, equivalent RIF levels were observed in both strains however, TMP was detected only in mc²155 cells, hence proving a positive correlation between potency and compound levels. To the best of our knowledge this is the first time LC-MS method has been used to measure compound levels in mycobacterial cells. We propose it to be a valuable tool to understand the lack of potency or resistance mechanisms in antimycobacterial drug development.

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation.

The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

Role of activation-induced deaminase protein kinase A phosphorylation sites in Ig gene conversion and somatic hypermutation.

Activation-induced deaminase (AID) is thought to initiate somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR) by the transcription-coupled deamination of cytosine residues in Ig genes. Phosphorylation of AID by protein kinase A (PKA) and subsequent interaction of AID with replication protein A (RPA) have been proposed to play important roles in allowing AID to deaminate DNA during transcription. Serine 38 (S38) of mouse AID is phosphorylated in vivo and lies in a consensus target site for PKA, and mutation of this residue interferes with CSR and SHM. In this study, we demonstrate that S38 in mouse and chicken AID is phosphorylated in chicken DT40 cells and is required for efficient GCV and SHM in these cells. Paradoxically, zebra fish AID, which lacks a serine at the position corresponding to S38, has previously been shown to be active for CSR and we demonstrate that it is active for GCV/SHM. Aspartate 44 (D44) of zebra fish AID has been proposed to compensate for the absence of the S38 phosphorylation site but we demonstrate that mutation of D44 has no effect on GCV/SHM. Some features of zebra fish AID other than D44 might compensate for the absence of S38. Alternatively, the zebra fish protein might function in a manner that is independent of PKA and RPA in DT40 cells, raising the possibility that, under some circumstances, AID mediates efficient Ig gene diversification without the assistance of RPA.

Mobilization of RAG-generated signal ends by transposition and insertion in vivo.

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

New concepts in the regulation of an ancient reaction: transposition by RAG1/RAG2.

The lymphoid-specific factors, recombination-activating gene 1 (RAG1) and RAG2, initiate V(D)J recombination by introducing DNA double-stand breaks at specific sites in the genome. In addition to this critical endonuclease activity, the RAG proteins catalyze other chemical reactions that can affect the outcome of V(D)J recombination, one of which is transposition. While the transposition activity of the RAG proteins is thought to have been critical for the evolution of modern antigen-receptor loci, it has also been proposed to contribute to chromosomal translocations and lymphoid malignancy. A major challenge has been to determine how the transposition activity of the RAG proteins is regulated in vivo. Although a variety of mechanisms have been suggested by recent studies, a clear resolution of this issue remains elusive.

DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase.

In addition to their essential role in V(D)J recombination, the RAG proteins function as a transposase capable of inserting the V(D)J recombination intermediate, the signal end DNA fragment, into target DNA. RAG-mediated transposition has been suggested to contribute to genome instability and the development of lymphoid malignancies. Previous studies suggested that the RAG transposase exhibits a target site preference for GC rich sequences and hairpin structures. Here we demonstrate that a transposition hot spot (5'-GCCGCCGGGCC-3'), smaller portions of this hot spot and other GC rich motifs are able to target RAG-mediated transposition. Tracks of GC base pairs have been shown to have an unusually high rate of base pair breathing. Intriguingly, we find that DNA mismatches can efficiently target RAG-mediated transposition and suppress the use of other target sites. Hairpins, however, are not generally preferred targets. Our results indicate that target DNA melting may be a crucial step during RAG-mediated transposition, and that target site selection by the RAG transposase may be intimately linked to mutagenic and metabolic processes that transiently present favorable DNA structures to the transposition machinery.

Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents.

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl- N-nitro- N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.

A hairpin near the 5' end stabilises the DNA gyrase mRNA in Mycobacterium smegmatis.

RNA is amongst the most labile macromolecules present in the cells. The steady-state levels of mRNA are regulated both at the stages of synthesis and degradation. Recent work in Escherichia coli suggests that controlling the rate of degradation is as important as the process of synthesis. The stability of mRNA is probably more important in slow- growing organisms like mycobacteria. Here, we present our analysis of the cis elements that determine the stability of the DNA gyrase message in Mycobacterium smegmatis. The message appears to be stabilised by a structure close to its 5' end. The effect is especially pronounced in a nutrient-depleted state. These results largely parallel the model proposed in E.coli for mRNA degradation/ stability with subtle differences. Furthermore, these results suggest that the slow-growing organisms might use stable mRNAs as a method to reduce the load of transcription on the cell.

DNA gyrase genes in Mycobacterium tuberculosis: a single operon driven by multiple promoters.

The two genes encoding DNA gyrase in Mycobacterium tuberculosis are present next to each other in the genome, with gyrB upstream of gyrA. We show that the primary transcript is dicistronic. However, in addition to the principal promoter, there are multiple weaker promoters that appear to fine-tune transcription. With these and other mycobacterial promoters, we propose consensus promoter sequences for two distinct sigma factors. In addition to this, the gyr genes in M. tuberculosis, as in other species, are subject to autoregulation, albeit with slower kinetics, probably reflecting the slower metabolism of the organism.

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase.

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase-DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. GyrI reduces intrinsic as well as toxin-stabilized gyrase-DNA covalent complexes. Furthermore, GyrI reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, GyrI is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.