Development of Inhibitors against Mycobacterium abscessus tRNA (m1G37) Methyltransferase (TrmD) Using Fragment-Based Approaches.

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures.

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 Å resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

The Escherichia coli biotin regulatory system: a transcriptional switch.

The ability of any organism to survive depends, in part, on mechanisms that enable it to modify its patterns of gene expression in response to extra- and intracellular signals. In the classical response mechanisms, a small molecule signal impinges on either an extra- or intracellular receptor, and through a series of events the signal is ultimately transmitted to transcription regulatory proteins. An alternative to this classical mechanism is provided by multi-functional transcription factors. These proteins function directly in transcription as well as in at least one additional cellular process. An example of this class of proteins includes the dimerization cofactor of hepatocyte nuclear factor (DcoH), which serves as an enzyme involved in regeneration of the tetra-hydrobiopterin cofactor and as a factor that stabilizes the dimerization of the hepatocyte nuclear transcription factor (Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, et al. Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Science 1991;254:1762-7; Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR. Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc Natl Acad Sci U S A 1992;89:11891-4). Another example is the protein PutA, a redox enzyme involved in proline utilization and a regulator of transcription of the genes involved in proline utilization (Ostrovsky de Spicer P, Maloy S. Puta protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci U S A 1993;90:4295-8). While several proteins of this class have been identified, their mechanisms of functional switching remain to be elucidated.