1. In future research, the optimization of ligands could be explored more thoroughly to in-
crease inhibitor specificity and potency. Most notably, further laboratory investigation
will needed to validate the applications and confirm the effects of the potential inhibitors
identified in this study. In parallel, the SpoT complex should be explored in detail in fu-
ture research since the reciprocal effect of the synthase-OFF hydrolase-ON confirmation
is extremely understudied. Furthermore, if these ligands are validated as potent inhibitors
of RelA-mediated biofilm synthesis, a novel branch of antibacterial agents may arise and
the integration of biofilm inhibitors with current antibiotics could yield a significant ad-
vancement in the field medicine.
The molecular docking studies utilized in this study have yielded several potential selective inhibitors of the RelA complex.
Interactions with key RelA residues are suggested to be pertinent to high ligand efficiency and strong binding energy. This
study reveals that benzene substituents are highly effective for refining ligand specificity and increasing binding affinity for
RelA residues HIS176, SER247, and ARG327. Additionally, RelA and ATPase are suggested by this study to have similar ac-
tive pockets; this is suggested by comparable active pocket volumes and ligand-residue interactions. Additionally, binding
energies were calculated for ATPase to ensure that the binding energies were three orders of magnitude lower for ATPase
than RelA. This preliminary measure suggests that adverse side effects could sufficiently be controlled in a dose-dependent
manner. In order to further increase the difference between RelA and ATPase binding energies, polar groups were explored as
substituents. Namely, functional groups, such as nitro groups (-NO2) and hydroxymethyl groups (-CH2OH), were explored as
viable mechanisms to block vital residue interactions on ATPase. Below (Figure 5) depicts the top-tier of selective ligand in-
hibitors prior to polar group insertion or modification. Ligand 2 is predicted to have the strongest binding energy with the
RelA complex and a difference of nearly 4 orders of magnitude when compared respectively to the binding energy of
ATPase. This ligand suffers from extremely low solubility (Sw = 0.0013 ug/mL). Ligand 1, exceeds the threshold of a three
orders of magnitude difference; however, solubility (Sw = 0.0013 mg/mL) still restricts its effectiveness as a drug candidate.
Thus, exploration of polar group additions and functional group substitutions will require further evaluation aimed to opti-
mize ligand properties vital to potential therapeutic applications. Ligand 5, despite its lower difference in binding energy
magnitude, is a potential candidate due to its solubility (Sw = 0.0034mg/mL). Although restrictive, Ligand 1 and Ligand 5
may serve as the starting ground for this study’s future ligand optimization and development.
Table 1: Tabulated data from molecular docking evaluations. Binding energies are given for the receptor
ATPase and RelA. Differences in binding energies denote separation in orders of magnitude.
Figure 5: Visual representation of Lig-
and 2 in preferred binding confirmation
with RelA. All close-contacts, polar
interactions, and hydrogen bonds are
labeled with the corresponding RelA
residue.
Figure 6: Visual representation of Ligand
5 in preferred binding confirmation with
RelA. All close-contacts, polar interac-
tions, and hydrogen bonds are labeled
with the corresponding RelA residue.
This study has primarily focused on inhibiting biofilm synthesis by disrupting RelA’s abil-
ity to identify hostile environmental conditions. The RelA-SpoT complex is a prime thera-
peutic target for increasing the efficiency of antibacterial agents. Despite the limited
knowledgebase on the RelA-SpoT complex, the accessibility of a crystal structure is an ex-
tremely valuable asset in this study. This crystal structure has enabled computer-assisted
molecular screening and facilitated the identification of key residues vital to the detection
of biofilm inducers, such as insufficient amino acids or antibacterial agents. This study sug-
gests that HIS176, SER247, and ARG327 are vital to RelA biofilm synthase activity. In
conjunction, this study was unable to explore the modes of SpoT activity due to limitations
of the crystal structure. Theoretically, inhibition of the RelA active site could activate SpoT
hydrolase activity. This would not only prevent biofilm formation but also promote the
breakdown of any residual biofilm components. The molecular docking evaluations pre-
formed in this study yielded several viable ligands. Of these, two possess adequate solubili-
ty values to be pursued as potential drug candidates. While continuation of this study will
be essential to validate and optimize these leads, as well as potentially identify new lead
compounds, this study has successfully supported the location and residues involved in ac-
tivating biofilm synthase activity, gathered knowledge on RelA’s pocket and interactions,
and proposed several ligands to be evaluated as potential drug candidates.
I’d like to offer a special thank you to the following people:
Dr. Haifeng (Frank) Ji and the Chemistry Department for their genuine passion for teaching and
research,
The Office of Undergraduate Research for affording me this opportunity and their dedication to
the STAR program,
The Cahill Family for supporting my education and career goals,
Drexel’s Freshman Rowing Team and Coaches for allowing me to pursue my goals to their fullest,
And everyone that was indirectly involved in my research and education.