Browsing by Author "McCafferty, Dewey G"

Natural products belonging to the lipid II-binding family act as potent antimicrobial agents by disrupting cell wall biosynthesis via sequestering the late-stage intermediate lipid II. However, the emergence of resistance mechanisms and poor bioavailability have hindered the utility of these molecules as promising therapeutic intervention strategies to combat pathogenic bacterial infections. Gaining a deeper understanding of structural components and biosynthetic pathways can lead to the creation of second-generation derivatives to improve bioactivity and pharmacological properties. To explore this superfamily, we have used bioanalytical, biochemical, synthetic, computational, and enzymatic approaches that have been applied to three distinct projects. The first includes efforts to characterize the relationship between structural feature and bioactivity for the lipid II-binding CDA (calcium dependent antibiotic), malacidin. Through a series of minimally complex analogs, we determined non-proteinogenic amino acids and the N-acyl fatty acid moiety are essential for bioactivity. For the second project, we investigated a conserved mechanism of action for phylogenetically-related natural products within the lasso peptide subfamily. This work led to the discovery of a novel class I lasso peptide, arcumycin, and we validated a conserved mechanism of action for Actinobacteria-produced lasso peptides in targeting lipid II biosynthesis. Our last project sought to elucidate the mechanism of lipoinitiation for the ramoplanin family of molecules. Through a series of bioactivity assays, we found the transfer to the acyl carrier protein (ACP) in a fatty acyl-AMP ligase (FAAL)-dependent manner determined the specificity of lipids selected in the biosynthetic process. Collectively, through each project we have gained a deeper understanding of the structural elements and biosynthetic pathways of lipid II-binding antimicrobials.

Parkinson's disease (PD) is a debilitating neurodegenerative disease of the central nervous system characterized by loss of striatal dopaminergic projections from the substantia nigra. Although there is no known cure for PD, dopamine (DA) replacement using L-3,4-dihydroxyphenylalanine (L-DOPA) is the most common therapy used to manage PD symptoms. L-DOPA is poorly absorbed into the brain and metabolized in the periphery causing its efficacy to wane with time. Additionally, within five years of use, L-DOPA can induce its own severe motor dysfunction, including dyskinesias, which can be irreversible. This underscores the need for the discovery and development of improved anti-parkinsonian therapeutics. We have identified a class of conformationally-constrained phenylethylamines based on a tranylcypromine scaffold and demonstrated that many compounds in this structural class exhibited partial or full relief of akinesia in a DA-deficient/DA transporter knockout (DAT-KO) mouse model of PD developed in the Caron laboratory. Two highly active arylcyclopropylamines from studies in DAT-KO mice were subsequently evaluated in a 6-hydroxydopamine-lesioned rat model to confirm their anti-parkinsonian and anti-dyskinesia activities. In these rats, both compounds improved lesioned-induced motor deficits that emulate akinesia. Target identification and activity assays suggest 5-HT2B and 2A as candidate targets to begin elucidation of novel non-dopaminergic pathways to combat PD.

Parkinson’s disease (PD), a common neurodegenerative disorder, can result from defective proteostasis mechanisms that induce neuronal toxicity. A common pathological hallmark of PD is the disruption of protein transport and trafficking between organelles within the neuron. To date, there has been some success in identifying small drug-like molecules that can restore defective trafficking pathways. These compounds are often identified through phenotypic screening in cellular models of PD toxicity, but, for many, the specific target of the compound and its mechanism of action are not fully understood. One such compound, identified in a screening effort in the laboratory of Susan Lindquist, was recently shown to alleviate multiple phenotypic markers of PD toxicity. In particular, the compound was shown to alleviate markers of toxicity induced by aggregation of α-synuclein, a protein that is genetically linked to PD. The activity of this compound, which contains a characteristic N-arylbenzdiimidazole (NAB) scaffold, was shown to depend upon E3 ubiquitin ligase Nedd4. Nedd4 has been previously implicated in PD toxicity as it regulates α-synuclein aggregation and proteostasis through ubiquitin signaling, but the mechanism of NAB compounds as modulators of Nedd4 was not elucidated. To this end, a series of biochemical and biophysical analyses were employed to understand the binding and mechanism of NAB2, the most potent NAB derivative, as a ligand of Nedd4. These experiments revealed that NAB2 binds to Nedd4 with high apparent affinity in the nanomolar range but does not induce changes in Nedd4 enzymatic activity in vitro. As Nedd4 activity is dependent upon protein-protein interaction and is tightly regulated at a cellular level, a proteomics-based evaluation of ubiquitination was pursued to study the effect of NAB2 treatment on the global ubiquitylome. This analysis revealed that induction of α-synuclein toxicity dramatically remodels the ubiquitylome, and NAB2 treatment induces small but phenotypically relevant changes in protein ubiquitination. Through this effort, a previously unrecognized Nedd4 substrate, trafficking scaffold protein TFG, was identified to be ubiquitinated in a NAB-dependent manner.

To further expand our understanding of the NAB2 mechanism in alleviation of α-synuclein toxicity, an unbiased chemoproteomic approach was employed to identify NAB2 targets across the proteome. This effort revealed small GTPase Rab1a, a regulator of endoplasmic reticulum (ER)-to-Golgi body transport, as an additional putative target of the NAB scaffold. This result is particularly promising as ER-to-Golgi transport is disrupted in PD toxicity and restored by NAB2 treatment. Further analysis of NAB2/Rab1a binding indicate that NAB2 binding occurs in a nucleotide-dependent manner, and NAB2 treatment phenocopies Rab1a overexpression by improving the viability of α-synuclein toxic cells. While the functional link between Nedd4 and Rab1a is not yet clear, the efforts toward understanding the NAB mechanism of action have revealed a protein network involved in NAB2-dependent rescue of trafficking defects and PD toxicity. Cumulatively, these results expand our understanding, at a molecular level, of PD toxicity and small molecule rescue thereof, enabling future efforts to target these proteins for development or optimization of neuroprotective compounds.

Antibiotic  resistance  is  a  crisis  in  modern  society  causing  increasing  rates  of   bacterial  infections  impervious  to  current  therapies.    To  this  end,  new  targets  for   antimicrobial  treatment  must  be  pursued.    Peptidoglycan  recycling  is  an  understudied   key  life  process  in  the  bacterial  cell  where  over  60%  of  cell  wall  materials  are  reused   during  each  turnover.    Anhydro-­N-acetylmuramic  acid  kinase,  AnmK  is  a  novel  enzyme   in  this  pathway  catalyzing  the  conversion  of  anhydro-­N-acetylmuramic  acid  to  N-acetylacetylmuramic  acid-­6-­phosphate  in  the  presence  of  magnesium  and  ATP.    Previously,   several  crystal  structures  of  AnmK  have  been  solved  providing  insights  into  the  catalytic   mechanism,  but  until  this  point,  no  extensive  work  has  been  done.    The  goal  of  this  work   is  to  determine  the  chemical  and  kinetic  mechanisms  of  AnmK.    This  will  be  completed   using  a  continuous  assay  for  dual  hydrolysis  and  phosphorylation  activity  as  well  as  a   novel  assay  for  carbohydrate  hydrolysis.    Substrate  interactions  will  be  probed  using   previous  crystal  structures  as  a  guide.    Finally,  pre-steady-­state  studies  will  conclude  the   mechanistic  studies  giving  a  full  depiction  of  AnmK  catalysis.  

The  kinetic  and  chemical  mechanisms  of  AnmK  are  studied  in  the  steady-­state   using  a  continuous  assay  format  where  bisubstrate  kinetics  as  well  as  inhibitor  studies   were  performed  as  well  as  substrate  specificity  studies.    pH  rate  profiles  and  solvent   isotope  exchange  were  used  to  determine  the  residues  involved  in  catalysis.      

The  concerted  or  stepwise  nature  of  hydrolysis  and  phosphorylation  is  a  main   question  of  this  work.    A  novel  assay  using  glucose  oxidase  was  executed  to  trap  any   chemical  intermediates  formed  in  a  potential  stepwise  reaction.    This  assay  was  used  on wild-type  AnmK  as  well  as  a  variety  of  mutants.    Through  these  experiments,  two  key   residues  in  phosphoryl  transfer  are  identified  and  used  to  partially  decouple  hydrolysis   and  phosphorylation.  

Mechanistic  studies  were  continued  by  investigating  the  pre-­steady-­state  kinetics   of  AnmK  using  a  quench  flow  apparatus.    Wild-type  AnmK  showed  no  appearance  of  a   chemical  intermediate  during  timepoints  as  short  as  10  ms  and  also  showed  a  linear   formation  of  product  with  a  catalytic  rate  analogous  to  the  steady-­state  rate.    These   results  indicate  that  AnmK  undergoes  a  concerted,  one-step  catalytic  mechanism  with   no  chemical  intermediate.    AnmK  E330A  formed  both  hydrolysis  product,  as  well  as   hydrolysis  and  phosphorylation  product  in  the  pre-steady-­state  agreeing  with  the   previous  results  that  hydrolysis  and  phosphorylation  had  been  partially  decoupled.  

These  results  show  the  chemical  and  kinetic  mechanism  of  a  novel  enzyme  with   previously  undocumented  concomitant  hydrolysis  and  phosphorylation.    This  work   provides  further  understanding  of  carbohydrate-modifying  enzymes  as  well  as  the   peptidoglycan  recycling  pathway.    A  better  understanding  of  peptidoglycan   biosynthesis  and  recycling  could  lead  to  novel  antimicrobial  therapies  in  the  future.

Histone modifications play important roles in regulating gene expression in various cellular processes by altering the underlying chromatin structure and thus influencing related pathological conditions. Histone methylation is one such modification that was thought to be static and enzymatically irreversible until the recent discovery of histone demethylases. Lysine specific demethylase 1 (LSD1) is a unique histone demethylase that removes methyl groups on lysine residues and mediates expression of many genes important in cancer progression. LSD1 activity and its substrate specificity are mainly regulated by association with a number of co-regulatory proteins. CoREST is one such important binding protein that endows LSD1 with the ability to associate with and demethylate nucleosomal substrates.

Given the significance of CoREST in directing LSD1 activity, herein we report our efforts to regulate LSD1 enzymatic activity by modulating its binding interaction with CoREST in order to provide a new means to inhibit the demethylation activity of LSD1 and thereby suggest a novel therapeutic intervention for breast cancer where LSD1 is implicated. Towards this end, initial steps have been taken to elucidate the molecular basis of the binding interaction between LSD1 and the functional region of CoREST, denoted as CoREST286-482 , by conducting isothermal titration calorimetry (ITC) studies. We found that the proteins tightly interact in a 1:1 stoichiometric ratio with a dissociation binding constant (Kd) of 15.9 ± 2.07 nM, and that their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Furthermore, we defined that the linker region of CoREST286-482 is a key determinant for the tight binding interaction toward LSD1. Accordingly, the peptide corresponding to the linker region of CoREST286-482 (designated as the Linker peptide) was used as a potent competitive modulator of the binding interaction between LSD1 and CoREST286-482 with our expectation that the Linker peptide will compete with CoREST286-482 for binding to LSD1 and thus sequester LSD1 from nucleosomal contexts to inhibit its demethylation activity.

Indeed, the Linker peptide has shown successful inhibitory activity against the binding of LSD1 to CoREST286-482 at nanomolar concentration. We evaluated the potential of the Linker peptide to inhibit LSD1 demethylation activity in cellular models of estrogen receptor a (ERa)-positive breast cancer cell line (MCF7) where LSD1 is known to co-localize with ERa and affect ERa-transcription activities. We were able to observe that the disruption of the binding interaction between LSD1 and CoREST by the Linker peptide affects not only the demethylation activity of LSD1 on both H3K4 and H3K9 but also the ERa-recruitment to promoters of target genes and processes required for proliferation of breast cancer cells. Thus it is believed that LSD1 demethylation activity through its physical and functional interaction with CoREST is essential in the development of ERa-positive breast cancer. Furthermore, we have observed that not LSD1 but CoREST physically interacts with ERa, which suggests a potential role of CoREST as a bridge connecting LSD1 to ERa. At the moment although the precise mechanisms underlying the implication of LSD1 through CoREST interaction in ERa-positive breast cancer still remain elusive, our study has verified that modulating LSD1 enzymatic activity by interrupting a critical protein-protein interaction offers a distinctively new avenue of inhibition of LSD1 activity, which has anti-cancer potential.

The obligated intracellular bacterium Chlamydia trachomatis is a pathogen of immense clinical and societal importance with over 1.5 million cases of new infection reported annually in the United States alone. With asymptomatic infection and no acquired immunity, Chlamydia infection often lead to epithelial scaring, ectopic pregnancy and infertility. Worldwide, the pathogen also responsibles for the ocular infection called trachoma that is the leading cause of infectious blindness. The bacteria have a diminutive genome consist of just over 900 genes. In addition, C. trachomatis had been historically refractory toward genetic manipulation. With the bacteria relied upon the host for basic metabolic resources, it is hypothesized that the genes retained in its genome is essential for the infectivity, pathogenesis and life cycle of bacteria. Through bioinformatics tools like sequence similarity networks, we have found that Chlamydia retained a complete set of genes involved in the biosynthesis of menaquinone, a membrane bound electron shuttle essential for cellular respiration. The biosynthesis of menaquinone in C. trachomatis involve the recently discovered futalosine pathway. In this project, we developed a molecular toolkit to examine the individual Chlamydial genes involved in menaquinone biosynthesis for its enzymatic function and essentiality. We employed small molecule inhibitors to chemically probe the function of genes in a cellular infection model and found that certain inhibitors of the menaquinone biosynthesis are effective at inducing inhibitory phenotype. Next, we developed a library of codon-optimized expression plasmid for recombinant expression of the Chlamydial protein in E. coli. We were able to enzymatically characterize and structurally determine the flavin prenyltransferase protein CT220 and its involvement in menaquinone biosynthesis. We also utilized E. coli mutants to test the function of the Chlamydial genes using genetic complementation. Lastly, we were able to improve the processes for plasmid-based mutagenesis of C. trachomatis using the recently developed fluorescent reported allelic exchange mutagenesis tool.

Lysine specific demethylase 1 (LSD1) is a FAD-dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor α (ERα), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2-arylcyclopropylamines as irreversible mechanism-based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ERα-postive and ERα-negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis.

Furthermore, we observe that LSD1 and ERα work cooperatively to express certain estrogen-target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ERα recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ERα, we believe that CoREST may be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST in vitro in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity.

In the fight against antimicrobial resistance, chemotherapeutic agents derived from natural products have served as a first line of defense. However, widespread antibiotic resistance has created an urgency for the development of new therapeutics. Ramoplanins and enduracidins are first generation nonribosomally-encoded lipodepsipeptides with potent activities against a broad spectrum of Gram-positive pathogens, including those resistant to front line antibiotics. As antimicrobial agents with exciting therapeutic potentials, strategies are warranted to develop access to second generation derivatives to improve drug stability and tolerability. To this end, a targeted genome mining strategy was devised to identify peptide congeners from sequenced bacterial genomes. We identified six biosynthetic gene clusters predicted to produce unique antimicrobial congeners. Two such peptides have been isolated from their native producing strains, Micromonospora chersina strain DSM 44151 and Amycolatopsis orientalis strain DSM 40040, and characterized to expand this class of antibiotics for the first time since the discovery of ramoplanin 30 years ago. We additionally have pursued multidisciplinary strategies to define the activity and selectivity of biosynthetic machinery. Together, this work has provided access to novel peptide scaffolds for further therapeutic development and establishes a platform for antimicrobial discovery and biosynthetic engineering of complex peptide derivatives.

Chlamydia trachomatis is the obligate intracellular pathogen responsible for the most common bacterial sexually transmitted infection worldwide. While our front-line antibiotics have been historically successful in combatting chlamydial infections, emerging issues including treatment failure and chlamydial persistence necessitate the development of new therapeutic approaches. In this work, an enzyme-focused approach was devised to explore two of the intricate survival strategies of C. trachomatis: virulence and energy metabolism. We sought to employ biochemistry, enzymology, and chemical biology tools to interrogate enzyme functions and inform the design of new antichlamydial agents. To these ends, our efforts focused on characterization of the virulence-associated effector protein chlamydial protease-like activity factor (CPAF) and the futalosine pathway for menaquinone biosynthesis. Mechanistic analyses of CPAF zymogen maturation and peptide hydrolysis were performed that collectively classified CPAF as a serine protease with a catalytic tetrad. Analogs of the natural product salinosporamide A were subsequently explored as CPAF inhibitors. The futalosine pathway was discovered to be a source of novel antichlamydial targets through traditional and chemical genetics analyses in a HeLa cell model of chlamydial infection. The foundation was also established for studying a specific pathway enzyme, CT263, in a continuous coupled enzyme assay. Collectively, this dissertation has progressed our knowledge of several enzymes involved in critical processes for chlamydial pathogenicity and viability. The insights gained on a mechanistic level and in the context of chlamydial infection have laid the groundwork for pursuing virulence and energy metabolism enzymes as antichlamydial targets and for developing inhibitors of their activity, which are much-needed resources to combat this extremely prevalent sexually transmitted infection.

During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease chlamydial protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Understanding the role of CPAF in pathogenesis is hampered because Chlamydia are not genetically tractable organisms. As such, chemical biology approaches were used to confirm CPAF function in vitro and in vivo, and to validate it as a virulence target. Here we report the development of assays, investigation of substrate specificity, and establishment of CPAF as a central virulence factor in chlamydial pathogenesis. A system for the expression and purification of CPAF was developed. An in vitro assay would allow for determination of kinetic parameters and aid in understanding the function of this protease. Two in vitro proteolysis assays, a discontinuous HPLC-based assay and a continuous fluorescence quenching assay, were developed for use in kinetic parameter determination and inhibitor discovery.

CPAF substrate specificity studies were conducted through the use of alanine scanning, proteomic identification of protease cleavage sites (PICS), and quantitative proteomics. Results from these studies showed that CPAF exhibited a preference for glycine, alanine, and serine in position P1, and valine in position P2' of peptide substrates.

Additionally, we designed and synthesized a zymogen-derived inhibitor peptide with nanomolar affinity that inhibited CPAF activity in vitro and in vivo. Using this, anti-CPAF peptide, we established CPAF as a virulence factor for chlamydial pathogenesis. Furthermore, CPAF inhibition resulted in degradation of the inclusion vacuole, exposing the bacteria and stimulating bacterial killing, thus CPAF inhibition created an antibacterial effect. CPAF inhibition also leads to the stimulation of innate immune defense activation, namely activation of caspase 1. In addition, CPAF was determined to be inhibited by the natural product salinosporamide A, a variant of omuralide, and the active form of the proteasome inhibitor lactacystin. Salinosporamide A and omuralide offer advantages over peptide therapeutics because of their intrinsic resistance to proteolytic degradation and improved oral bioavailability. Toward that end, progress toward CPAF inhibitor derivates from this natural product scaffold is also presented. Collectively this thesis lends support for CPAF as an antivirulence target for Chlamydia.

Bacteria recycle pre-existing peptidoglycan in order to minimize the de novo synthesis of peptidoglycan precursors. The recycling pathway is under study for its chemotherapeutic target potential. Anhydromuramic acid kinase (AnmK) is part of this recycling pathway and catalyzes the dual hydrolysis/phosphorylation of anhMurNAc to MurNAc-6-P. This enzyme has been discovered and introduced, but only minimally characterized. Therefore, the overarching goal of this work was to clone, express and purify AnmK to homogeneity; perform further kinetic characterization; solve the open, closed and transition state mimic-bound conformations of AnmK by x-ray crystallography; and develop a putative mechanism based on the accumulated research findings and 18O-labeling studies.

The anmK gene was successfully cloned as a hexahistidine fusion protein and overexpression was optimized. After exhaustive trials, a final purification scheme was designed to yield homogeneous AnmK in three chromatographic steps and in less than 36 hours. Additionally, the synthesis of both anhMurNAc and a pseudosubstrate (anhGlcNAc) were carried out in 35% and 77% overall yield, respectively. The synthesis of these compounds allowed for both kinetic characterization and structural studies.

To this end, the structure of de novo AnmK was solved using SAD and high-resolution (1.9 Å) data. Also, an ATP analog (ANP) and anhMurNAc substrate-bound, closed conformation structure (1.95 Å) was solved. These structures elucidated an 11° domain closure of the enzyme upon substrate binding and also revealed the active site geometry to be used to determine potential molecular determinants of specificity.

Insights into the kinetic mechanism of AnmK were then gathered using multiple techniques. First, the structure of AnmK (2.5 Å) was solved the with a known transition state analog, the MgADP-vanadate complex. Following this structure, which sheds light on the potential importance of a residue other than the catalytic base (Asp187), isotopic labeling was performed with H₂18O. Using NMR and MS, the regiochemical selectivity of AnmK hydrolysis to impart the solvent derived oxygen at C1 was established. Additionally, this was carried out with stereochemical preference to create the α-anomer of the carbohydrate product. This regiochemistry and stereospecificity drove the design of our putative concomitant hydrolysis/phosphorylation mechanism but we are not able to rule out the formation of a transient phosphoenzyme intermediate.

This research can be applied to the immediate goal of understanding the function of a single, novel enzyme with unique chemistry and the clarification of the AnmK mechanism will facilitate future investigation into enzymes with dual hydrolase/kinase functionality. Furthermore, this research contributes to understanding of the complex bacterial peptidoglycan layer in order to harness new ideas for developing antibiotics.

In the last two decades, bacterial infections have reemerged as a serious public health threat. In the United States, nosocomial infections alone cost several billion dollars and are responsible for 100,000 deaths annually.3 Of particular concern is the emergence of bacteria with single and multidrug resistance. Infection by such bacteria can be difficult, if not impossible, to treat and cure. In recent years, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most threatening resistant strains. MRSA infections are now responsible for more deaths each year than AIDS in the United States.4 Due to the multidrug resistance of these "superbugs", new antibiotics with novel mechanisms of action are urgently needed.5

Unfortunately, although novel antibiotics are needed to stem the resistance crisis, the pipeline for novel antibiotics has narrowed considerably in recent decades.3,6,7 In the last thirty years, only the oxazolidinones and cyclic lipopeptides have been developed as novel antibiotic classes.7 One novel cyclic lipodepsipeptide antibiotic in the clinical pipeline is WAP-8294A2. WAP-8294A2 (Figure 1.0.4) was isolated from the soil bacteria Lysobacter staphylocidin (FERM BP-4900) in Japan in 1997. It is the major component of a mixture of eighteen related compounds, all of which showed antibiotic activity.13 WAP-8294A2 showed strong in vitro antibiotic activity against Gram-positive bacteria including vancomycin-resistant Enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) (Table 1.1).14,15 In vivo assays also showed that WAP-8294A2 has strong activity against known MRSA strains.13 We were interested in gaining synthetic access to WAP-8294A2 and analogs to establish a system for structure-activity relationship (SAR) studies. We also sought to further characterize the proposed mechanism of action and identify and characterize the biosynthetic machinery.

We developed a solid-phase strategy for the development of WAP-8294A2 and analogs with on-resin cyclization based on molecular modeling studies. We chose a macrocyclization site between Glu8 and Asn9 to take advantage of a predicted turn element and used the carboxylate side chain of glutamic acid as a convenient handle for resin attachment. We describe three analogs that were synthesized and analyzed for their biological and mechanistic profiles, providing evidence that the N-acyl fatty acid tail and the depsipeptide ester bond are required for antibiotic activity, but not for binding to the membrane phospholipid cardiolipin.

Additionally, we developed a method to improve the production, isolation, and purification of the WAP-8294A antibiotic complex highly enriched in the A2 fraction. The antibiotic complex was analyzed for antibiotic activity and was found to be equally active to the reported values for the A2 fraction. Basic mechanism of action studies with cardiolipin confirmed the ability of the WAP-8294A mixture to bind to cardiolipin and also suggested that the binding is mediated through interactions with the phosphate groups in the polar headgroup of cardiolipin.

Finally, we sequenced the genome of L. staphylocidin and inspected it for the WAP-8294A biosynthetic machinery. We successfully identified the WAP-8294A NRPS biosynthetic gene cluster in L. staphylocidin. It is composed of two large open reading frames (ORFs) that encode NRPS machinery made up of 44 domains organized into 12 modules. The organization of the gene cluster argues for a co-linear assembly of the peptide template. We also identified the genes flanking the large NRPS ORFs, which encode for a redox enzyme and several host regulation and preservation proteins.

In the course of examining the L. staphylocidin genome for the WAP-8294A biosynthetic gene cluster, we discovered a single module hybrid PKS/NRPS with high homology to heat-stable antifungal factor (HSAF). HSAF has been identified in two strains of L. enzymogenes, but was previously unknown in L. staphylocidin. We identified the genes in this cluster and compared them to the previously known HSAF biosynthetic gene clusters noting important similarities and differences between them.

The obligate intracellular pathogen, Chlamydia trachomatis, is becoming an ever greater public health threat worldwide. Despite aggressive public health awareness campaigns and treatment with antibiotics, chlamydial infections continue to be the most frequently reported sexually transmitted infection in the United States and the cause of 3% of worldwide blindness. While research into understanding various mechanisms of chlamydial pathogenesis is ongoing, efforts to identify critical protein targets are hampered by the lack of facile genetic manipulation systems available for Chlamydia. Without the ability to perform genetic studies, researchers have employed chemical biology tools to close the gap in understanding how Chlamydia survives and thrives in the host cell.

Chlamydial protease-like activity factor (CPAF) has been identified as a central virulence factor in chlamydial pathogenesis. Several studies have indicated a role for CPAF-mediated degradation of host proteins in the late stages of infection. CPAF is hypothesized to interfere with myriad host cell processes, including inflammation, cell proliferation, cytoskeletal development, and immunity presentation. However, recent studies have called into question the methods used to previously identify bona fide in vivo CPAF targets, as CPAF has been shown to retain proteolytic activity even in the presence of broad spectrum protease inhibitors. As a result of these new finding, there is a renewed call to carefully identify CPAF substrates using methods that ensure total inhibition of post-lysis proteolysis.

This dissertation aims to clarify the role of CPAF in chlamydial pathogenesis and to identify mechanisms by which CPAF exhibits substrate specificity. Because enzymes can manifest specificity through kinetic mechanisms, sequence recognition, secondary site substrate binding, or protein structure level specificity, multiple methods of biochemical characterization were employed to distinguish between these modes of specificity.

Optimized HPLC-based and fluorescence quenching assays were developed and used to investigate the chemical and kinetic mechanism of CPAF proteolysis, as well as to characterize CPAF resistance to broad spectrum protease inhibitors. Peptide library proteomics were designed to probe active site sequence recognition of specific amino acids. Bioinformatic approaches were used to recognize and annotate a cryptic PDZ-like domain in CPAF, which bears strong structural similarity to human epithelial tight junction proteins. Using a new endocervical cellular model of infection, a recently developed C. trachomatis mutant lacking CPAF activity was investigated. Mass spectrometry proteomics analysis was employed to detect differential cleavage of host proteins in endocervical cells infected with CPAF+ and CPAF- strains of C. trachomatis. Lastly, methods for N-terminal labeling and enrichment were adapted for further identifying CPAF substrates in a cellular infection model. The subtiligase system for biotinylation of N-terminal amines was adapted for integration with C. trachomatis infection assays and downstream mass spectrometry proteomics. Ultimately, the dissertation offers clarification of the role of CPAF in chlamydial infection and provides chemical biology tools for further study of protease function in bacterial pathogenesis.

The posttranslation modification of histone proteins within the nucleosomes of chromatin plays important roles in the regulation of gene expression in both normal biological and pathobiological processes. These modifications alter local chromatin structure and subsequently alter the expression profile of associated genes. Histone methylation, which was long thought immutable, is one such modification that plays a dual functionality in both activation and repression of gene expression and can be thought of as an information storage mark. With the initial discovery of lysine-specific demethylase 1A (LSD1/KDM1A), an FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively, the missing counterbalance to dynamic histone methylation was cemented. This discovery further strengthened the link between histone demethylation and transcriptional regulation and the enzyme has since been identified as a target with therapeutic potential.

Given the significance of KDM1A enzymatic activity, herein we report our efforts to characterize novel binding interactions that dictate the enzymes biological and pathobiological functions. As KDM1A falls into the greater class of flavin-dependent amine oxidases, it contains features that are recurrent within the class, but due to its unique ability to work on histone and non-histone substrates has unprecedented structural elements. Although the active site is expanded compared to the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint of the histone H3 tail. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. Using steady-state kinetic analyses, we now show that unmodified histone H3 is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM that is approximately 100-fold higher than the 21-mer peptide product. The relative affinity of dose-response curves is independent of preincubation time suggesting that H3 rapidly reaches equilibrium with KDM1A. Rapid dilution experiments confirmed the increased binding affinity of full-length H3 toward KDM1A was at least partially caused by a slow off-rate with a koff of 0.072 min-1, a half-life (t1/2) of 9.63 min, and residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction revealing a Kd of 9.02 ± 2.27 nM, a kon of 9.26 x 104 ± 1.5 x 104 M-1s-1 and koff of 8.35 x 10-4 ± 3.4 x 105 s-1. Additionally, consistent with H3 being the only histone substrate of KDM1A, no other core histones are inhibitors of demethylation activity. Our data suggests that KDM1A contains a histone H3 secondary specificity recognition element on the enzyme surface and required further characterization.

In order to characterize this secondary H3 binding site, we turned to the use of cysteine labeling, chemical cross-linking coupled to proteolysis and LC-MS/MS, HDX-MS, and the design of an active, tower domain deletion KDM1A mutant. We now show that the tower domain contributes to the extended binding interface of the KDM1A/H3 interaction. Additionally, we show that the KDM1A tower domain is not required for demethylation activity and that one can functionally uncouple catalytic activity from protein-protein interactions that occur along the KDM1A tower domain interface, a domain unprecedented in the greater amine oxidase family. Furthermore, this towerless mutant will be useful for dissecting molecular contributions to KDM1A function along the tower domain. Our discovery of this secondary binding site within the aforementioned domain points to how pivotal this region is to the control and localization of KDM1A enzymatic activity as it also serves a pivotal role as a protein-protein interaction motif for the nucleation of a multitude of multimeric protein complexes.

With this in mind, we set out to design a strategy to isolate the core histone demethylase complex from E. coli cellular lysates. With the use of polycistronic vectors that encode both KDM1A and CoREST for coexpression we were able to produce appreciable amounts of chromatographically pure complex. As our CoREST construct in this strategy contains both the ELM2 and SANT2 domain needed for interaction with the HDACs, this core complex will serve as a starting point for future work that will tease apart additional influences on substrate binding and recognition imparted on KDM1A from binding partners. This preparation can therefore be used in a multitude of downstream studies including reconstitution of the core histone demethylase/deactylase complex and in depth kinetic and biophysical analyses and provides an invaluable starting point

This work provides a foundational understanding of this unprecedented secondary binding site on the surface of the KDM1A tower domain and how it may play an important role in substrate and product recognition. We suspect that this extended interaction interface may control KDM1A localization within specific chromatin loci and allow the enzyme to serve as a docking element for the nucleation of protein complexes or transcriptional machinery. On the other hand, disruption of this point of contact between the KDM1A/H3 binary complex may also facilitate enzyme/product dissociation, thereby tuning the catalytic activity of the demethylase. Additionally, the ability to produce substantial quantities of the core histone demethylase complex is a necessary step in the decoding of the ‘histone code’ hypothesis of KDM1A and its associated complexes. We suspect that the body of this work will prove to be invaluable for future characterization of the enzyme and its role in biology and pathobiology.

The loss of nigrostriatal dopaminergic neurons is the fundamental hallmark of Parkinson’s disease (PD). In early PD stages, this is ameliorated by dopamine (DA) supplementation; however, as the disease progresses, the complete loss of this key dopaminergic pathway forces the central nervous system to find alternative routes to regain motor control. It has previously been shown that serotonergic routes must take on the role of the failed dopaminergic system throughout the progression of the disease. Previously studied 5-HT1A anxiolytic and anti-depressive therapeutics have yet to be successfully repurposed for Parkinson’s disease patients. Herein is described the current efforts towards the employment non-dopaminergic agonists in the investigation of motor control in Parkinson’s disease. This research outlines the development of non-dopaminergic therapeutics inspired by the core structure of the clinically approved 5-HT1A agonist Befiradol. This motif has been infused with a trans-2-arylcyclopropylamine moiety which has been independently shown to reduce motor symptoms in Parkinson’s disease via a prior collaboration from the McCafferty lab. While it was originally hypothesized that these therapeutics would act as bifunctional agonists at the 5-HT1A and M4 GPCRs, affinity assays reveal dualistic agonism at the 5-HT1A and 1 receptors, offering a new class of potential bifunctional therapeutics.

The post translational modification (PTM) of histone proteins is a highly dynamic process that is utilized in the control of gene transcription. This epigenetic process involves enzymatic ‘writers’ and ‘erasers’ which place or remove chemical modifications to the unstructured tails of histone proteins which protrude out from the nucleosomal core. In a highly dynamic manner, each PTM is spatiotemporally regulated and combinations of PTMs at a gene promotor or enhancer region leads to transcriptional enhancement or repression. The gene targets as well as selectivity and specificity of epigenetic enzymes is regulated by the multimeric complexes each enzyme is co-opted. Each complex contains a unique set of coregulatory proteins with RNA and DNA binding domains and PTM ‘reader’ domains to direct the catalytic machinery to a specific subset of genes. The coregulatory proteins also affect the specificity and selectivity of the enzyme through mechanisms which are only beginning to be explored.

Our interest is in elucidating the role of coregulatory proteins and lncRNA with respect to lysine-specific demethylase 1 (LSD1/KDM1A). A flavin-dependent mono-and di-demethylase of H3K4me1/2 and H3K9me1/2, KDM1A has been implicated in many different multimeric enzymatic complexes which, in some cases such as the REST and NuRD complexes, function on opposing pathways. This disparity in the downstream outcome being coordinated by the same enzyme highlights the need to understand not only epigenetic enzymes, but to consider the complexes as a whole towards therapeutic targeting.

The specific aims of my thesis were to (a) interrogate the role of individual and multiple coregulatory partners in enzyme selectivity and specificity (b) establish tools to study the mechanisms of biochemical and biophysical of protein:protein and protein:lncRNA interactions and (c) elucidate key characteristics of protein:protein and protein:lncRNA interfaces towards targeted disruption. To this end, I have utilized cloning and mutagenesis methods to heterologously express and purify coregulatory partners of KDM1A in E. coli. I chose coregulatory partners found in a common catalytic core as well as several additional coregulatory proteins from a stable KDM1A-containing 5-mer complex. I have produced multiple constructs for four of these proteins to allow for multiple affinity purification routes as well as for future binding studies. I have further expressed each of these constructs and have made significant efforts towards the purification of each construct based on solubility.

I furthermore established HDX-MS and SELEX protocol in our lab as tools to allow us to explore the dynamics of these epigenetic interactions. I further demonstrated and confirmed that there is no hotspot along the binding interface between KDM1A and CoREST, but that CoREST stabilizes the apical end of the KDM1A tower domain via HDX-MS with the highest change in deuterium uptake, over 20%, long KDM1A TαA residues 440-451.

I also made significant efforts towards elucidating the interaction between KDM1A and HOTAIR. Firstly, I established an RNA radiolabeled EMSA assay for the lab which allowed us to test the binding of HOTAIR to KDM1A. With this assay, we saw that CoREST286-482, specifically the linker region (residues 293-380), must be bound to KDM1A for HOTAIR to bind and that the dissociation constant was unchanged at 1.710.38 µM and 1.29±0.34 µM, respectively. Further, I confirmed that the first 320 nt of domain 4 of HOTAIR (nt 1500-1820) contain the critical binding and that the dissociation constant was slightly higher at 2.97±0.96 µM.

I have also optimized SHAPE-MaP and crosslinking strategies to explore the binding interface between KDM1A:CoREST286-482 and lncRNA. I determined that there were 83 nt that displayed at least a 1.5-fold change in SHAPE reactivity of HOTAIR D4 due to the presence of KDM1A:CoREST286-482. I also utilized a free-energy based secondary structure model to establish a secondary structure for HOTAIR D4 based on my SHAPE-MaP data. I noted that 44% of the significant nt were confined to a stretch of RNA (nt 1538-1610, 1779-1844) that is predominantly dsRNA. Further usage of photochemical crosslinking strategies revealed a propensity for G:C paired nt to be crosslinked to KDM1A:CoREST286-482. A similar nt sequence around these paired nt suggests a binding motif. The implications of these results is discussed herein.

Gram-positive pathogens, such as Streptococcus pyogenes and Staphylococcus aureus, are etiological agents of a large array of human diseases. Unfortunately, our ability to treat these infections is increasingly limited due to the development of bacterial resistance to many existing therapies. Thus, novel targets for antimicrobial development are urgently needed. An attractive candidate for a new class of anti-virulence chemotherapeutics is the sortase class of enzymes. Sortases are extracellular transpeptidases unique to Gram-positive bacteria. Their function is to covalently attach secreted virulence factors to the bacterial cell wall. Deletion or inhibition of sortases results in severe attenuation of bacteria for infection. In order to develop novel effective antimicrobial agents, a robust understanding of the biological and chemical mechanisms of the target are required. To this end, this dissertation endeavors to further illuminate the biochemical mechanism of sortase enzymes and to extend the current knowledge of the roles of sortases and their substrates during infection.

Through steady-state kinetics, active site reactivity measurements, three-dimensional structure determination via X-ray crystallography, and computational modeling of substrate binding, the basic enzyme mechanism of S. pyogenes sortase A (SrtA) has been revealed. In general, S. pyogenes SrtA displays many of the same mechanistic characteristics as previously studied sortases, including a reverse protonation mechanism, a conserved tertiary structure arrangement, and utilization of similar substrate binding interfaces and conserved active site residue functions. These findings suggest a general sortase mechanism, conserved among classes and species.

Initial steps have also been taken to characterize S. pyogenes sortase C (SrtC). SrtC enzymes are unique in that they covalently polymerize secreted proteins, rather than attach them to peptidoglycan. Full length and truncation mutant constructs of SrtC and its substrate, T3, and peptide substrate mimics have been produced in soluble form for use in kinetic assays. Additionally, initial crystallization conditions have been identified for S. pyogenes SrtC towards the goal of three-dimensional structure determination. A homology model of the structure has also been produced, displaying many of the general features observed for other sortase enzymes.

Additionally, a computational analysis of the mechanism of isopeptide bond formation in S. pyogenes SPy0128, a substrate of S. pyogenes SrtC, has been performed. Isopeptide bonds have previously been found in structural studies of Gram-positive bacterial adhesins in each domain of these multi-domain proteins. The bonds are typically formed between conserved lysine and asparagine residues, and formation is likely catalyzed by adjacent conserved glutamates. A direct nucleophilic attack mechanism, starting from an inverse protonation state, is supported in this study. Of note, there appears to be temporal regulation of isopeptide bond formation in the different domains of S. pyogenes SPy0128, with the C-terminal domain isopeptide bond forming prior to or simultaneously with the N-terminal domain isopeptide bond.

Previous studies suggest that SrtA activity is required for S. aureus to survive phagocytosis by a macrophage. The production of reactive oxygen species by professional phagocytes could lead to inhibition of SrtA via oxidation of a conserved nucleophilic cysteine residue in the active site. Through determination of inhibition kinetics, identification of oxidative modifications, reduction potential measurements, and analyses of SrtA in vivo activity in the presence of reactive oxygen species, it has been demonstrated that S. aureus SrtA is resistant to oxidative inhibition. These findings support SrtA activity inside the phagolysosome of a professional phagocyte and likely contribute to the ability of S. aureus to evade the innate immune system.

The roles of sortases and their substrates during S. aureus survival inside professional phagocytes have not been thoroughly investigated. Through analysis of the regulation of these surface proteins under phagolysosomal conditions and macrophage phagocytosis survival assays, initial characterization of the functions of sortases and their substrates in this environment has been completed. Previous studies have suggested a role for SrtA and its substrate, Protein A, and these genes and two other sortase-substrates were upregulated in response to phagolysosomal conditions. However, neither sortases nor their substrates demonstrated a direct function in phagocytosis survival. These findings imply a complex interplay between S. aureus and professional phagocytes. Further studies are necessary to delineate the direct activities of surface anchored proteins during phagocytosis of S. aureus by professional phagocytes.

The natural progression from the introduction of an antibiotic into the market to the emergence of resistant strains demands the constant influx of new drugs to treat infection. Ramoplanin A2, a drug with demonstrated resistance against antibiotic-resistant Gram-positive pathogens, is currently in clinical trials. Ramoplanin is composed of seventeen amino acids condensed with a N-acylated fatty acid tail and a sugar moiety. The biosynthesis of this important metabolite is performed by a host of enzymes including non-ribosomal peptides synthetases, fatty acid biosynthetic enzymes, glycosyltransferases, and other tailoring enzymes.

In the introductory chapter, the significance and mechanism of the family of ramoplanin antibiotics will be discussed. The biosynthetic cluster of the lipoglycodepsipeptide ramoplanin and its sister antibiotic enduracidin will be outlined and the previous work on this cluster summarized. The proposed formation of the critical N-acylated tail and the parallels to fatty acid biosynthesis will also be described in detail. Chapter two describes the development of an expression system to successfully isolate proteins of interest from the ramoplanin biosynthetic cluster. In addition to increasing the yields of proteins already expressing in heterologous hosts, this technique facilitated the isolation of previously unattainable proteins. This chapter also details the demonstration of their activity through a variety of biochemical techniques.

Chapter three details the kinetic characterization of Ramo16, a NAD-dependent β-ketoacyl reductase, critical for the formation of the N-acylated fatty acid chain attached to Ramoplanin. Sequence analysis and characterization of the product of the Ramo16 reaction also implies that the biosynthesis of the N-acylated tail of ramoplanin bears significant similarity to type II bacterial fatty acid biosynthesis. These observations have led to a new proposal for the biosynthesis of the N-acylated fatty acid attached to ramoplanin. Further supporting these conclusions, chapter four details the examination of the stereochemistry of the hydrogen removed from the nicotinamide adenine dinucleotide cofactor and the stereochemistry of the product of the enzymatic reaction.

Chapter five describes the 1.4 Å crystal structure of ramoplanin in the presence of CTAB, an amphipathic membrane phospholipid mimetic. Observation of the location of the N-acylated fatty acid allows interpretation of its critical role in the activity of ramoplanin. The formation of the dimer and the interactions between the two molecules of ramoplanin and ramoplanin and the surroundings were examined in detail and supported earlier structure activity relationships. These observations have led to a new model for ramoplanin and Lipid II recognition in the context of the bacterial membrane. Enduracidin, ramoplanin's sister antibiotic, is produced by S. fungicidicus. Development of fermentation conditions for this strain allowed the isolation of appreciable amounts of enduracidin and 15N-labeled enduracidin, which was used to examine enduracidin's binding to Park's nucleotide, a cell wall precursor to Lipid II, by 2D 1H- 15N HSQC NMR. Although chemical shifts in the HSQC spectrum indicated binding, insoluble fibril formation complicated interpretation and necessitated the use of crystallography to determine the interaction with the substrate. Initial crystal conditions have been determined and diffraction images have been collected at 3.0 Å. Higher resolution images are pending to determine this structure.

Finally, chapter six discusses the development of a technique to transform S. fungicidicus and introduce plasmids to obtain deletion and insertional disruption mutants. A high throughput PCR screen was determined to enable large batch screening of potential mutants. This technique may be applied to study the function of genes in the pathway by fermenting production of enduracidin and examining the results.

Lysine-specific demethylase 1 (LSD1/KDM1A) regulates transcriptional events by post-translational modifications of histone H3 tails at residues K4 an K9. This enzyme plays a vast number of roles in both normal cellular functions and diseases states. Increasingly it is appreciated that this enzyme, like most epigenetic regulators, does not function alone, but rather forms a catalytic subunit of much larger protein assemblies that congregate on chromatin to concertedly mediate transcriptional events. LSD1 in particular has been found in many different complexes, in many different tissues and can facilitate both activation and repression events.

Because of these roles, LSD1 is viewed as a potential therapeutic target. Significant effort has recently led to the development of highly selective and potent active-site inhibitors. These inhibitors have particularly shed light on the cancer-promoting activities of LSD1 in acute myeloid leukemia and small cell lung carcinoma. However, one failing of these strategies is that active site inhibition is incapable of differentiating between the multitude of functions LSD1 performs. We sought to address this issue by instead developing first-generation tools to explore protein-protein interaction disruption as an alternative strategy for inhibiting the enzyme.

To this end, we have carefully examined a well-characterized interaction between LSD1 and the scaffolding protein CoREST. Using this interaction as a template, we developed a probe we show can compete with CoREST for interaction with LSD1. Furthermore, we generated cell permeable versions of this probe and examined the effects in a model of breast cancer. We find that our probe can selectively inhibit estrogen signaling, a feat that was not possible with current small molecule inhibition or RNA interference technologies. We therefore we propose that disrupting interactions such as this is an excellent alternative for targeting “undruggable” proteins, but also may also expand the current therapeutic space by granting precise control over the individual functions of proteins.