By Sloan Grissom, Kate Webster,

Ruthie Stewart & Caroline Plymel

Purpose and Relevance of research
● Purpose: To develop a new antibiotic to treat pathogenic bacteria by inhibiting bacterial
RNA Polymerase:
○ Bacteria can develop resistance to antibiotics relatively quickly due to selective
pressures
○ Existing RNAP inhibitors largely only target gram positive bacteria
● Relevance: Gives health professionals more and more effective strategies to fight
bacterial infections, which is especially important in life threatening cases or in cases
where bacteria is already resistant to one class of drugs
○ Example: Methicillin Resistant Staphylococcus aureus (MRSA) is a Staph infection
that can be treated effectively by very few drugs right now
What we already know
● RNAP targeting drugs have already been proven effective against TB and biofilm
infections, but largely only work on gram positive bacteria
○ There’s a need for RNAP antibiotics that treat gram negative bacteria
● Bacteria reproduce quickly, so drug resistance can develop rapidly and become a big
problem
● Some bacteria have already developed resistance to RNAP inhibiting drugs, so new
strategies for interfering with RNA polymerase are being tested
○ Targeting different binding sites on the enzyme
○ Finding non-competitive antibiotics (they don’t compete with each other)
● Many antibiotics we use now come from natural sources (i.e. are secondary metabolites
of bacteria), but developing compounds to specifically treat infections may prove more
effective
What’s unclear and what’s being done about it
● Why current RNAP inhibiting antibacterials aren’t as effective against gram negative
bacteria
○ Researchers for this paper used E. coli, a gram negative bacteria, in their
experiments to try and develop effective RNAP inhibiting drugs from gram
negatives
○ Some of the same researchers published an earlier paper also using E. coli in their
experiment to identify a natural RNAP inhibitor of gram negatives, Salinamide F
● Why certain bacteria have developed resistance rifamycins (the current RNAP inhibiting
class of drugs) so quickly
○ The resistance has already developed, so the only way to solve the problem is to
create different antibiotics
What’s new
● In the past, the majority of antibacterial drugs have come from secondary metabolites of
microorganisms
○ While these drugs have been effective, we have a limited amount of them simply
because microorganisms only make a few specific secondary metabolites that are
useful to us
● In this research a compound known as MRL-436 is used to inhibit RNAP activity
○ The origins of MRL-436 aren’t specifically stated in the paper, but it’s termed a
“novel small molecule”
○ Hasn’t been used before because it was recently identified as a potential
antibacterial agent in unpublished research
MRL-436
Hypotheses
● MRL-436 is effective in inhibiting RNA polymerase activity in E. coli
● MRL-436 is effective in inhibiting RNA polymerase activity in mutant strains of
E. coli resistant to rifamycins because it binds to a different site on RNAP
● MRL-436 resistant strains of E. coli are not resistant to rifamycins because of
different binding sites
Methods - Affinity Selection Mass Spectrometry (AS-MS)
● Assesses the binding of molecules to biomolecular targets
● Steps:
1. Affinity Selection Stage
a. Protein to compound
2. Receptor-Ligand Complexes are separated from non-binding
components
3. Ligands are identified using mass spectrometry
Mass Spectrometry
● Ionizes chemical species and separates based on mass-to-charge ratio
● Separates through electronic or magnetic field
● Ions with same mass-to-charge ratio will undergo the same amount of
deflection
● Detected by a machine such as an electron multiplier
● Displayed as a spectra showing the relative abundance
● Identify sample with known atomic masses
Liquid Chromatography - Mass Spectrometry
● LC: method of separation in which the compounds of a liquid mixture are
distributed between two immiscible phases (stationary and mobile).

Types
● Adsorption chromatography
● Partition chromatography
● Ion-Exchange chromatography
● Size-Exclusion chromatography
● Affinity chromatography
Automated Ligand Identification System (ALIS)
● Dual chromatography LC-MS system that incorporates size exclusion with
reversed-phase LC-MS
● Size-exclusion to separate complexes from unbound constituents and detect
binding compounds.
● Steps:
a. Affinity Selection
■ Target protein mixed with
mass-encoded library and
physiologically relevant
buffer
b. Separation
c. Reverse-Phase Chromatography
d. Ligand Detection

https://www.researchgate.net/figure/222704926_fig1_Fig-1-Diagram-of-the-Automated-Ligand-Identification-System-ALIS-affinity
In this paper...
● RNA core enzyme, RNAP holoenzyme were prepared from E.coli
● Screened 43,000 molecules that supposedly have growth-inhibitory activity
against a sensitized E. coli strain by binding ability to RNA Polymerase
● Bound molecules were tested as a single, pure compound against the RNAP
target and against a negative control protein to rule out nonspecific binding
● Then, separated and identified protein-bound compounds using
reversed-phase LC-MS
● Compounds that showed reproducible MS signals specific to the RNAP target
were considered to be hits
● Use a selective hit (MRL-436) that exhibited concentration dependent binding
Does MRL-436 inhibit RNAP?
● Fluorescence-detected
● DNA fragment containing
lacUV5 promoter as template
and profluorescent analog
● Ribogreen exhibits
fluorescence upon binding to
RNA
Results
MRL-436 Binds to E. Coli bacterial RNA
polymerase (RNAP)
•Does not compete with Rif for binding to RNAP
•Inhibits RNAP
•RNAP-inhibitory activity is responsible for its
antibacterial activity
MRL-436 binds to E. Coli RNAP
Affinity Selection – Mass Spectrometry (AS-MS)
•To identify molecules that would bind to E. Coli RNAP
–Over 200 identified
–MRL-436 – one of 14 that reproducibly and selectively bound to
(RNAP)
•To perform RNAP competition binding assays
 Table 2 A
Normalized AS-MS response of Rif and MRL-436 to increasing
concentrations of MRL-436

MRL-436: Increasing concentrations

•Rif: fixed concentration
MRL-436 exhibits clear
concentration-dependent binding to RNAP
Table 2 B
Normalized AS-MS response of Rif and MRL-436 to increasing
concentrations of Rif

•Rif: increasing concentrations

•MRL-436: fixed concentrations
Binding site on RNAP for MRL-436 is
different from, and does not overlap,
binding site for Rif on RNAP
Table 2 C
Inhibition of RNAP by MRL-436 & Rif
•Transcription assays -
determine whether MRL-436
inhibits RNAP
•Results - MRL-436 shows
concentration-dependent
inhibition of RNAP
MRL-436 inhibits RNAP
Effects of MRL-436 on Wild-Type RNAP
& on Rif-Resistant Derivatives
•Transcription assays used to compare effects
•Rif-resistant RNAP derivatives
–Contain amino-acid substitution in Rif binding sites
–Substitutions interfere with Rif binding
–Selected for analysis at substitution at three sites most frequently substituted in
Rif-resistant isolates
•Results – MRL-436 is fully effective against Rif-resistant derivatives
Table 1
MRL-436 Inhibition of RNAP, Including Rif-Resistant
RNAPs

•MRL-436 inhibits Rif-resistant RNAP

•Supports results that binding sites on RNAP for
MRL-436 & Rifampin do not overlap
Inhibition of RNA synthesis by MRL-436
in bacterial cells
•Show that antibacterial activity of MRL-436 was due to its RNAP-inhibitory activity
•Assessed effects of MRL-436 on E. Coli strain after incorporations of:
–Thymidine into DNA
–Uridine into RNA
–Amino acids into protein
Figure 3
Inhibition of RNA synthesis by MRL-436 in bacterial cells
•Left: MRL-436 data
•Right: Rif data
•Black: Absence of inhibitor
•Red/Blue: Presence of inhibitor
•Asterisks: Statistically significant
differences (t test; p < 0.02)
Figure 3 Results
MRL-436
•Inhibits:
–RNA synthesis from earliest time point after addition
–Protein synthesis only at late points after addition
•Does not inhibit DNA synthesis at any time point
•Selectively inhibits RNAP synthesis in bacterial cells
•Supports original hypothesis
 Discussion
Research objective:

● The goal of the research was to identify a novel antibacterial agent that inhibits RNAP
through a binding site that does not overlap the rifamycin binding site and that does not
share the cross resistance with rifamycins

In conclusion…

● MRL-436 is a novel, small molecule that binds to and inhibits E. coli RNAP.
● MRL-436 does not compete with the bacterial RNAP inhibitor Rifamycin for binding to
RNAP because of different binding sites.
● Therefore, E. coli may be resistant to MRL-436 or Rifamycin alone, but it is unlikely that E.
coli will be resistant to both.
● This may lead to the creation of antimicrobials that prevent antibiotic resistance.
 Strengths and Limitations of the Study
● Strengths:
○ Used two approaches to test whether RNAP inhibitory activity of MRL-436 is responsible for its
antibacterial activity.
■ MRL-436 resistant mutants
■ Effects of MRL-436 on incorporation of labeled amino acids into proteins, thymidine into
DNA, and Uridine into RNA.
○ 100% accuracy when concluding that all MRL-436 resistant mutants had mutations in RNAP
genes
● Weaknesses:
○ Limited to E. coli.
○ Does MRl-436 compete with other commonly used RNAP inhibitors used as antibiotics?
■ Results may change when studying other antibiotics because MRL-436 and the
antibiotic may have overlapping binding sites.
○ When studying the resistant mutants the sample size was only 6.
 MRL-436 vs. Rifamycin
● Why do we need both Rifamycin and MRL-436?
● MRL-436 can kill rifamycin resistant E.coli and Rifamycin can kill bacteria
resistant to MRL-436.
○ How is this possible when both antibacterials work using the same mechanism: RNAP
inhibition?
● A single amino acid substitution of RNAP 622 beta residue or deletion of the
RNAP omega subunit makes the E. coli resistant to MRL-436 but not to
Rifamycin.
● Both RNAP beta 622 residue and RNAP omega subunit are far from the
binding site for Rifamycin.
Rifamycin &
MRL-436
● Figure 4. shows the
separate binding
sites of Rif and
MRL-436.
 Cellular Alarmone vs. MRL-436
● Previous research has shown that RNAP beta residue 622 and RNAP omega
subunit have been shown to form part of a binding site on RNAP for a cellular
alarmone. This may result in RNAP inhibition by the alarmone.
○ A Cellular alarmone is an intracellular signal molecule produced because of harsh
environmental factors that regulates gene expression at transcription level.
● Because both the cellular alarmone and MRL-435 are affected by the
mutation of the RNAP beta and omega region, this indicates that the binding
site for the alarmone and the binding site of MRL-436 are probably similar.
● However, this has not yet been proven through experimentation and further
research needs to be done.
 Ethical Considerations
● Once the inhibitors of bacterial RNAP were found, this study looked for and cultured
bacteria that was resistant to this molecule, MRL-436.

● They then found the specific area on the gene and the mechanism behind these
bacteria’s resistance.
○ i.e. RNAP b 622 residue and RNAP w subunit

● Therefore, this study's technique increases researcher’s knowledge of the mechanism

behind bacterial resistance, which is very beneficial for health care.

● However, this could be used to create a microbe resistant to many types of

antimicrobials and could be a tool in bioterrorism.
 Future Work in RNAP Inhibitors
● By finding RNA inhibitors that do not overlap, you decrease the likelihood of
antibiotic resistance.
● This is because the inhibitors bind to separate sites requiring mutations in
these binding sites to induce resistance.
● Combining RNAP inhibitors into one antimicrobial would require two separate
mutations in both the different binding sites which is statistically unlikely.
● Therefore, creating antimicrobials that include multiple RNAP inhibitors, one
may decrease the chances of resistance substantially.
● More research with different antibiotics that function as RNAP inhibitors.
 Bibliography
Hassan, H.M., Degen, D., Jang, K.H., Ebright, R.H., and Fenical, W. (2015). Salinamide F, new depsipeptide antibiotic and
inhibitor of bacterial polymerase from a marine-derived Streptomyces sp. National Institute of Health, 68, 206-209.

Howe, J.A., Want, H., Fischmann, T.O., Balibar, C.J., Xiao, L., Galgoci, A.M., . . . Rigden, D. (2015). Selective
small-molecule inhibition of an RNA structural element. Nature, 526, 627-677.

Walker, S.S., Degen, D., Nickbarg, E., Carr, D., Soriano, A., Mandal, M., . . . Young, K. (2017). Affinity selection- mass
spectrometry identifies a novel antibacterial RNA polymerase inhibitor. ACS Chemical Biology.

Gross, M. L., Chen, G., & Pramanik, B. N. (2012). Protein and peptide mass spectrometry in drug discovery. Hoboken, NJ:
Wiley.

https://www.medicinescomplete.com/mc/rem/2012/c19-fig-19-6.png
Questions?