Atefeh Garzan

Repurposing antipsychotic drugs into antifungal agents: Synergistic combinations of azoles and bromperidol derivatives in the treatment of various fungal infections

As the number of hospitalized and immunocompromised patients continues to rise, invasive fungal infections, such as invasive candidiasis and aspergillosis, threaten the life of millions of patients every year. The azole antifungals are currently the most prescribed drugs clinically that display broad-spectrum antifungal activity and excellent oral bioavailability. Yet, the azole antifungals have their own limitations and are unable to meet the challenges associated with increasing fungal infections and the accompanied development of resistance against azoles. Exploring combination therapy that involves the current azoles and another drug has been shown to be a promising strategy. Haloperidol and its derivative, bromperidol, were originally discovered as antipsychotics. Herein, we synthesize and report a series of bromperidol derivatives and their synergistic antifungal interactions in combination with a variety of current azole antifungals against a wide panel of fungal pathogens. We further select two representative combinations and confirm the antifungal synergy by performing time-kill assays. Furthermore, we evaluate the ability of selected combinations to destroy fungal biofilm. Finally, we perform mammalian cytotoxicity assays with the representative combinations against three mammalian cell lines.

Enzymatic Evidence for a Revised Congocidine Biosynthetic Pathway

Naturally produced pyrrolamides, such as congocidine, are nonribosomal peptides that bind to the minor groove of DNA. Efforts to delineate the biosynthetic machinery responsible for their assembly have mainly employed genetic methods, and the enzymes responsible for their biosynthesis remain largely uncharacterized. We report the biochemical characterization of four proteins involved in congocidine formation: the adenylation‐thiolation (A–T) di‐domain Cgc18(1–610), its MbtH‐like partner SAMR0548, the AMP‐binding enzyme Cgc3*, and the T domain Cgc19. We assayed the ATP‐dependent activation of various commercially available and chemically synthesized compounds with Cgc18(1–610) and Cgc3*. We report the revised substrate specificities of Cgc18(1–610) and Cgc3*, and loading of 4‐acetamidopyrrole‐2‐carboxylic acid onto Cgc19. Based on these biochemical studies, we suggest a revised congocidine biosynthetic pathway.

Combating Enhanced Intracellular Survival (Eis)-Mediated Kanamycin Resistance of Mycobacterium tuberculosis by Novel Pyrrolo[1,5-a]pyrazine-Based Eis Inhibitors.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure-activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.

Deciphering Nature’s Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(A3aM3SA3bT3) and TioN(AaMNAb). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis.

Activation and Loading of the Starter Unit during Thiocoraline Biosynthesis

The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for AMP activation and carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by tioJ knockout and in vitro activation of 3HQA studies. However, we demonstrated that TioJ-activated 3HQA is not loaded onto the T domain TioO, as originally believed, but instead onto a fatty acid synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located outside of the thiocoraline gene cluster. We showed a strong interaction between TioJ and FabC. By generating TioJ point mutants mimicking the active site of highly homologous enzymes activating different molecules, we showed that the identity of the substrate activated by adenylation domains such as TioJ is not determined by only the active site residues that directly interact with the substrate. The insights gained from these enzymatic transformations are valuable in the efforts toward deciphering the complete biosynthetic pathway of thiocoraline and bisintercalators in general.

Interfering With DNA Decondensation as a Strategy Against Mycobacteria

Tuberculosis is once again a major global threat, leading to more than 1 million deaths each year. Treatment options for tuberculosis patients are limited, expensive and characterized by severe side effects, especially in the case of multidrug-resistant forms. Uncovering novel vulnerabilities of the pathogen is crucial to generate new therapeutic strategies. Using high resolution microscopy techniques, we discovered one such vulnerability of Mycobacterium tuberculosis. We demonstrate that the DNA of M. tuberculosis can condense under stressful conditions such as starvation and antibiotic treatment. The DNA condensation is reversible and specific for viable bacteria. Based on these observations, we hypothesized that blocking the recovery from the condensed state could weaken the bacteria. We showed that after inducing DNA condensation, and subsequent blocking of acetylation of DNA binding proteins, the DNA localization in the bacteria is altered. Importantly under these conditions, Mycobacterium smegmatis did not replicate and its survival was significantly reduced. Our work demonstrates that agents that block recovery from the condensed state of the nucleoid can be exploited as antibiotic. The combination of fusidic acid and inhibition of acetylation of DNA binding proteins, via the Eis enzyme, potentiate the efficacy of fusidic acid by 10 and the Eis inhibitor to 1,000-fold. Hence, we propose that successive treatment with antibiotics and drugs interfering with recovery from DNA condensation constitutes a novel approach for treatment of tuberculosis and related bacterial infections.