Assimilation of exogenous heme is compulsory for the prosperity of Pseudomonas aeruginosa, as well as other species of bacteria. In P. aeruginosa, two heme assimilation systems are essential - the Heme Assimilation System (Has) andPseudomonasHeme Utilization (Phu). HasA is a secreted hemophore that binds extracellular heme and brings it to HasR, a membrane receptor protein that incorporates heme into the cell. In parallel, PhuR is another membrane receptor protein that binds heme and brings it into the cell. Once heme gets internalized, PhuS then shuttles the assimilated heme to HemO, which is a heme oxygenase that breaks down heme into beta-biliverdin, carbon monoxide, and free iron that can be used for a multitude of intracellular processes. Therefore, heme assimilation systems serve as useful strategies for iron acquisition and have been proved to play essential roles in chronic P. aeruginosa infections in cystic fibrosis patients. The Boon lab has shown that NosP is a hemoprotein that senses environmental nitric oxide and labile heme to regulate the activity of a N osP-associated histidine kinase (NahK). In P. aeruginosa, our lab has shown that NosP/NahK signaling pathway is essential for sensing NO to trigger biofilm dispersal; however, it is still unknown if NosP/NahK signaling pathway also possesses additional functions of sensing assimilated heme to elicit downstream physiological responses, and whether the heme sensing properties of NosP/NahK are functionally dependent on Has and Phu systems.

Pyocyanin is a blue colored redox active phenazine, a major virulence factor. Its production is positively regulated by pseudomonas quorum sensing molecule, PQS. This can be correlated to data from our laboratory in PA14;  when nahK is knocked out, an increase  in pyocyanin and PQS levels are also observed. Since ∆nahK gives a similar phenotype to ∆ hemO, it is suggestive that the heme assimilation systems and/or HemO activity may be epistatic of the NosP/NahK signaling system. Heme assimilation is required by P. aeruginosa in chronic infections, therefore understanding and ultimately targeting heme sensing/assimilation pathways may help combat chronic pseudomonas infections.

Strokes are one of the leading causes of death worldwide, and ischemic strokes account for about 87% of strokes in the US and about 62% of strokes globally. Ischemic strokes are made more complicated by ischemic-reperfusion injury (IRI), in which cells begin to rapidly dysfunction and die during the restoration of blood flow to the ischemic tissue. In the heart and brain, IRI is exacerbated by dysregulation of the mitochondrial transition pore (mPTP). Cyclophilin D (CypD) is a peptidylprolyl isomerase and is a key modulator of the mPTP, where binding of CypD to the mPTP favors the open conformation of the pore. Inhibition of CypD lowers the probability of pore opening when there is substantial inorganic phosphate, Pi, present, suggesting CypD inhibition is a viable therapeutic target.

Currently, there is only one FDA-approved cyclophilin inhibitor, Cyclosporine A (CsA), which, while potent, is non-specific and binds to several cyclophilin isoforms, leading to unwanted side effects. Our group has previously shown that building compounds into the specificity pocket of the CypD active site is a promising approach to developing isoform-specific inhibitors, though these initial compounds had poor cell permeability.

To address this, we are using a fragment-based drug design approach in combination with computational docking to identify novel CypD inhibitors with  more desirable small molecule chemistries. We then validate the computational results with traditional biochemical and structural biological approaches. .

Karishma Ramcharan

M.S. in Chemistry, Stony Brook University, 2023
B.S. in Biochemistry, Hofstra University, 2021

Program/Dates Supported by NIGMS: Biochemistry/ August 2024 - present

Title: Regulation of the T3SS and T6SS through the NahK/HptB phosphorelay signal in Pseudomonas aeruginosa

Affecting more than 5.6 million people in the United States in 2020 and doubling in cases every 5 years for people over the age of 65, Alzheimer’s disease (AD) is one of several neurodegenerative diseases associated with the dysfunction of tau protein. In the central nervous system, tau is responsible for aiding in the development and stabilization of neuronal microtubules, which are critical to the integrity and function of axons. In AD and other forms of dementia, tau protein aggregates irreversibly in the presence of RNA and other biomolecules resulting in the deposition of “tau tangles”, a process  strongly correlated with disease progression. The mechanism by which this aggregation occurs remains poorly understood, impeding the development of therapeutic remedies for AD and other tauopathies.

Using light scattering experiments and coarse-grained molecular dynamics simulations, my project focuses on developing a self-consistent model for predicting the structural and thermodynamic properties of tau protein in solution. Taking into consideration the interplay between electrostatic, hydrophobic, and hydrogen bonding interactions, the model will predict tau behavior based on its primary sequence of amino acids and their associated side-chain bonding properties, such as their charges at physiological pH. Elucidating the effect of intermolecular interactions on the properties of tau protein, this model will provide a strong foundation for understanding greater mechanisms of aggregation, an important step towards the development of therapeutic agents for AD and other tauopathies.

Grace Yoon

B.S. in Chemistry, Stony Brook University

Program/Dates Supported by NIGMS: Chemistry/ June 2023-present

Urinary tract infections (UTIs) are one of the most common infectious diseases in both community and hospital settings cause primarily by the uropathogenic Escherichia coli (UPEC). Although standard antibiotic treatment is often successful in clearing UTIs there is a high rate of recurrence. In addition, there is emergence of multidrug resistant bacteria that represents a major health threat, for which new alternatives to traditional antibiotics are urgently needed. My research focuses on understanding the intricate molecular mechanisms utilized by the Gram-negative bacteria (UPEC) to coordinate the assembly and secretion of cell surface structures called pili. Pili serve as critical virulence factors, and the knowledge acquired from this research holds promise for the development of anti-virulence therapeutics. In my project, I have set out to employ the powerful technique of FRET (Förster resonance energy transfer) to probe and quantify specific interactions occurring at the bacterial outer membrane during pili assembly, with a specific focus on P pili, which play an important role in facilitating kidney colonization. I aim to acquire an understanding of how the usher selectively recognizes pilus subunits in the correct order, as well as the mechanisms underlying activation of the usher to initiate pilus biogenesis. This research project will elucidate fundamental mechanisms of protein translocation across membranes, the assembly of complex organelles, and virulence factor biogenesis at the bacterial outer membrane. Another facet of my project involves harnessing the power of FRET as a tool to screen small molecules, probing their potential to disrupt the process of pilus biogenesis. My project will facilitate the identification of novel anti-virulence compounds that inhibit assembly of adhesive pili by Gram-negative bacterial pathogens, selectively disrupting host-pathogen interactions required to initiate and sustain infection.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the second leading infectious killer after COVID-19. The rise of multi-drug- and extensively drug- resistant cases of TB reveal a pressing need for the development of new drugs against Mtb. Although an attractive drug target is the unique diderm envelope that harbors essential processes like uptake and export of metabolites, the mycobacterial cell wall proteome is not well-elucidated. In determining the proteome of the cell wall, we can identify these essential processes and, within these, potential drug targets. Our lab has successfully used proximity labeling with APEX2, an engineered peroxidase, to label proteins in a compartment-specific manner. To expand our understanding of the mycobacterial cell envelope, we have genetically encoded APEX2 to an outer membrane protein, MspA. Biotin-phenol, a commonly used APEX2 substrate, is cell permeable, labeling proteins in both the periplasm and cell wall. Considering this, we hope to synthesize a substrate that is unable to enter the cell. Here, we use the coupling reagent N-N’diisopropylcarbodiimide (DIC) to activate a carboxylic acid and react with a free amine to form an amide bond. 5-aminofluorescein (FAM) was chosen as the basis for the cell impermeable substrate because of its hydrophilicity and anionic charge at neutral pH. As a control, the starting material 7-hydroxycoumarin-3-carboxylic acid was chosen for its ability to cross the mycobacterial membrane. These products, FAM-phenol and 7-hydroxycourmarin-phenol, will hopefully help us cross the final frontier to understanding uptake and export at the mycobacterial cell surface.

Astrocytes are the most abundant non-neuronal cell type in the brain, providing support, nutrient buffering, and instructions to neighboring cells, particularly neurons, in order to maintain normal physiological function and homeostasis. Importantly, astrocytes play a large role in the brain by responding to insult, communicating with neurons via gliotransmitters, and forming protective barriers around endothelial cell layers. Because astrocytes are present abundantly in all cortical areas of the brain, performing essential metabolic, structural, and homeostatic roles, it is not surprising that they are implicated in a slew of disease and disorders, including, but not limited to, Alzheimer’s, Parkinson’s, glioblastoma, stroke, seizure, infection, and depression. Thus, it is becoming increasingly important to consider these cells for therapeutic intervention of such disease, seeing that they are important players in maintaining normal brain function. In leveraging organic cation transporters (OCTs) on the surface of astrocytes, we have developed a system by which a permanently positive N-heterocyclic amine can be attached, via organic chemistry, to diverse small molecules like fluorophores, photoactivatable markers, or calcium indicators and delivered specifically to astrocytes over neurons and other glia (Fig. 1a and b). These data have inspired us to think about more translational applications, including the potential to deliver drugs or transcription activators to astrocytes for therapeutic intervention. Gene-targeted therapies are currently in the clinic, but there exists a lack of regulated systems. We currently have some control over where, but no control over when therapeutic genes are expressed. I plan to synthesize and assay a doxycycline modified with a permanently positive N-heterocycle to exploit the powerful tetracycline-inducible gene expression system (Tet-On/Off) in order to control gene expression in astrocytes, exclusively. In adding this modification, I hypothesize that we will be able to target astrocytes for therapeutic intervention, decrease concentrations of drug needed by increasing blood brain barrier (BBB) penetration, and reduce off target effects of current constitutively active therapies as seen in Glial cell-derived neurotrophic factor (GDNF) or Neurturin (NTN) gene therapy for Parksinson’s disease.

Computer aided drug design is an integral part of modern drug discovery, particularly for hit identification. A hit is typically defined as a molecule which has a micromolar affinity for a protein target and is a starting point for optimization. This stage of drug development is particularly amenable to the use of computational techniques as they can search large areas of chemical space in a quick and efficient manner. My research aims to further improve this process via the development of new routines and algorithms in DOCK6. 

One such method for hit identification is the fragment by fragment construction of molecules within the constraints of a binding site, called de novo design. Currently DOCK6 can bias the construction of molecules towards those which are more similar to a given reference molecule, such as an experimentally validated inhibitor. This is achieved through the use of similarity-based scoring functions which assess the similarity of growing molecules to the reference molecule. Those which are most similar are advanced to subsequent stages of growth. However this inadvertently creates a bias for molecules which are bulkier at early stages of growth, and leads to ill-informed decisions. I believe that by reconstructing the reference molecule in parallel to the growth of new molecules, a higher degree of similarity can be achieved. This dynamic scoring will further focus the search for new hits in areas of chemical space which are likely to be successful, while still identifying novel chemical structures.

For successful viral replication to occur, viruses must be able to subvert host cell immune mechanisms. In Adenoviruses, a family of nonenveloped DNA viruses, the Adenovirus early protein E4ORF3, a SUMO E3 ligase, has been shown to inhibit host responses that could otherwise block viral replication.  Examples of targets with known functions include several involved in detecting and repairing double-strand DNA breaks, such as the Mre11-Rad50-Nbs1 (MRN) complex and p53. If not inhibited by the virus, these proteins can negatively impact Ad replication by responding to the virus’ double-stranded DNA genome as they would to a chromosomal double-strand DNA break. E4ORF3 also targets PML nuclear bodies, which are involved both in cell responses to DNA damage and in transcriptional regulation and antiviral responses such as the interferon response.  Another major target of E4ORF3 is the transcription factor TFII-I, which suggests that it is involved in suppressing Ad infection. TFII-I is involved with multiple transcription pathways, including some activated by p53 in response to DNA damage, and can inhibit the transcription of the Ad protein L4, which is important for the transition between early and late viral transcription in Ad infection.  Some studies have also associated TFII-I with the DNA Damage Response that the MRN complex functions in. However, its exact function in Ad infection is not known, and represents an important area of research to better understand the cellular mechanisms involved in Ad infection and possible general functions of TFII-I in viral infections. 

Our lab has already created TFII-I CRISPR knockouts in normal human bronchial epithelial cells (NHBEC), and Ad strains that lack E4ORF3. I am also currently developing TFII-I CRISPR knockouts in small airway epithelial cells (SAEC). The goal of my project is to investigate cellular antiviral responses to Ad infection and Ad expression of early and late viral proteins both with and without TFII-I and E4ORF3 expression.  Such investigation has the potential to increase our understanding of TFII-I as an anti-viral effector protein and of cellular mechanisms with the potential to block DNA viruses from successfully completing infection.