Staphylococcus aureus rnpa inhibitors: Computational-guided design, synthesis and initial biological evaluation

Article abstract of DOI:10.3390/antibiotics10040438

Antibiotic resistance is spreading worldwide and it has become one of the most important issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In Staphylococcus aureus, RnpA has been hypothesized to be one of the main players in these mech-anisms. S. aureus RnpA is able to modulate mRNA degradation and complex with a ribozyme (rnpB), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR) has only been partially explored. Accordingly, in the present work, using computational modeling of S. aureus RnpA we identified putative crucial interactions of known RnpA inhibitors, and we used this information to design, synthesize, and biologically assess new potential RnpA inhibitors. The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine of JC2, and the semithiocarbazide, the furan, and the i-propylphenyl ring of RNPA2000. Our efforts could provide a foundation for further computational-guided investigations.

Full text of DOI:10.3390/antibiotics10040438

antibiotics
Article
Staphylococcus aureus RnpA Inhibitors: Computational-Guided
Design, Synthesis and Initial Biological Evaluation
Lorenzo Suigo 1 , Michaelle Chojnacki 2, Carlo Zanotto 3, Victor Sebastián-Pérez 4, Carlo De Giuli Morghen 5,
Andrea Casiraghi 1, Paul M. Dunman 2, Ermanno Valoti 1 and Valentina Straniero 1,
*
1
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Luigi Mangiagalli 25,
20133 Milano, Italy; lorenzo.suigo@unimi.it (L.S.); a.casiraghi.89@gmail.com (A.C.);
ermanno.valoti@unimi.it (E.V.)
Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave.,
Rochester, NY 14642, USA; michaelle_chojnacki@urmc.Rochester.edu (M.C.);
paul_dunman@urmc.rochester.edu (P.M.D.)
2
3
4
5
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano,
Via Vanvitelli 32, 20129 Milano, Italy; carlo.zanotto@unimi.it
Exscientia, The Schrödinger Building, Oxford Science Park, Oxford OX4 4GE, UK;
victorsebastianperez@gmail.com
Department of Chemical—Pharmaceutical and Biomolecular Technologies, Catholic University “Our Lady of
Good Counsel”, Rr. Dritan Hoxha, 1025 Tirana, Albania; carlo.degiulimorghen@unimi.it
Correspondence: valentina.straniero@unimi.it; Tel.: +39-0250319361
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Antibiotic resistance is spreading worldwide and it has become one of the most important
issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery
are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In
Staphylococcus aureus, RnpA has been hypothesized to be one of the main players in these mech-
anisms. S. aureus RnpA is able to modulate mRNA degradation and complex with a ribozyme
(rnpB), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have
recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR)
has only been partially explored. Accordingly, in the present work, using computational modeling
of S. aureus RnpA we identified putative crucial interactions of known RnpA inhibitors, and we
used this information to design, synthesize, and biologically assess new potential RnpA inhibitors.
The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to
both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We
evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine
of JC2, and the semithiocarbazide, the furan, and the i-propylphenyl ring of RNPA2000. Our efforts
could provide a foundation for further computational-guided investigations.
Citation: Suigo, L.; Chojnacki, M.;
Zanotto, C.; Sebastián-Pérez, V.;
Morghen, C.D.G.; Casiraghi, A.;
Dunman, P.M.; Valoti, E.; Straniero, V.
Staphylococcus aureus RnpA Inhibitors:
Computational-Guided Design,
Synthesis and Initial Biological
Evaluation. Antibiotics 2021, 10, 438.
https://doi.org/10.3390/
antibiotics10040438
Academic Editor: Constantinos
Stathopoulos
Received: 12 March 2021
Accepted: 12 April 2021
Published: 14 April 2021
Keywords: antibiotic resistance; RnpA inhibitors; MRSA; UAMS-1; RNA degradation machinery;
antimicrobial activity; gram-positive bacteria
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
The well-documented consequences of antimicrobial resistance have been exacerbated
by the limited pharmaceutical investment in new molecules [
over-prescription and misuse [ ]. Indeed, antimicrobial resistance is currently surging in
every country of the world, as reported by the World Health Organization (WHO) [ ]. In
this context, the resistance of several pathogens, such as Klebsiella pneumoniae, Escherichia
coli, Mycobacterium tuberculosis, and Plasmodium falciparum, to both front line and antibiotics
of last resort, are progressively leading to higher incidences of morbidity and mortality,
1], together with antibiotic
2
Copyright:
© 2021 by the authors.
3
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
as well as increased healthcare costs that are related to prolonged stays in hospitals [
4].
Furthermore, because most of the commercially available antibiotics belong to the same
Antibiotics 2021, 10, 438. https://doi.org/10.3390/antibiotics10040438
https://www.mdpi.com/journal/antibiotics

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structural classes, the development of resistance to a single agent can rapidly broaden to
the entire structural class [5].
Among the pathogenic and highly virulent bacteria comprised in the ESKAPE clas-
sification (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), methicillin-resistant Staphylococ-
cus aureus is responsible for the majority of nosocomial infections worldwide [
associated with a 64% increase of death risk in comparison to the drug-sensitive strains [
In addition to methicillin, S. aureus has developed resistance to virtually all other front
line antibiotics, including vancomycin [ ], ceftaroline, linezolid, and daptomycin [ 10 14].
6
–8
], and it is
3
].
9
8,
–
These alarming data, together with a limited antibiotic development pipeline [15], necessi-
tate screening chemical space for agents that inhibit novel antimicrobial targets as a means
to develop new chemical classes of antimicrobials.
Post-transcriptional regulation has recently been recognized as a crucial process in
both eukaryotic [16] and prokaryotic cells for the control of gene expression [17–19]. In
S. aureus, mRNA turnover is able to finely regulate the transient expression of several
virulence factors, allowing for the bacterium to colonize the host [20]. In both E. coli and
S. aureus, the degradosome is also required for the maturation of several stable RNA species
(rRNA, tRNA) [17,21]. For these reasons, very recently, the enzymes catalyzing bacterial
RNA processing and degradation have emerged as potential targets for the development
of novel antimicrobials [20].
While in E. coli, the main player of the degradosome is the essential endoribonuclease
RNase E [17,20], S. aureus does not contain an RNase E ortholog, rather the 13 KDa
protein RnpA seems to contribute to cellular mRNA degradation as well as a second
RNA-metabolic process. Indeed, RnpA has been shown to catalyze the digestion of mRNA
and it can interact with the ribozyme rnpB, thus forming the RNase P riboprotein complex,
which is responsible for the removal of the 50 leader sequences from precursor tRNA
(ptRNA), thus promoting tRNA maturation [20]. As such, the protein may be a novel
antimicrobial target that is required for two essential processes, mRNA degradation and
ptRNA processing, and allows for the identification of agents that inhibit either one or both
processes. Eidem and co-workers developed and fully studied one of the first and most
promising RnpA inhibitors, RNPA2000 (Figure 1), which has been shown to inhibit RnpA
mediated mRNA turnover and ptRNA maturation [22].
Figure 1. Structure of RNPA2000, JC1, JC2, and JR1, known RnpA inhibitors.
Starting from RNPA2000, Lounsbury and co-workers developed a series of analogues
to overcome the structural weaknesses of this molecule, starting from the replacement

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of the 2-furanyl moiety [23]. In parallel, Colquhoun and colleagues discovered two ad-
ditional structural classes of potential RnpA inhibitors, the piperidine carboxamides and
phenylcarbamoyl cyclic thiophenes. Several compounds of these classes, such as JC2 and
JR1 (Figure 1), demonstrated the ability to interfere with both RnpA-mediated mRNA
degradation and ptRNA processing, with very low IC50 values and low mammalian
cell cytotoxicity.
JC2 was also evaluated in vivo using a Galleria melonella model of S. aureus infection,
and it proved to be able to reduce the mortality rate at 16 h to 30%, which was comparable to
the antibiotic vancomycin [24]. Yet, our understanding of the structure activity relationship
(SAR) from previously described RnpA inhibitors remains at its infancy.
Therefore, expanding the SAR of the RnpA inhibitors is the main aim of the present
work. Guided by a novel computational approach to predict the protein/compound
binding sites, we designed and synthesized 14 RNPA2000 or JC1
1–14) to probe key features of each chemical scaffold.
/JC2 derivatives (Figure 2,
Figure 2. Structures of compounds 1–14, object of the present work.
Compounds
1–
5
aimed to assess the importance of each single moiety of JC1/JC2
,
where compounds
1–3 bear, in lieu of the 3,5-dichlorophenyl scaffold, a biphenylic one.
As a result, we could evaluate the importance of the phenylic ring and, at the same time,
we evaluated whether a bulkier, more lipophilic, and electron rich substituent could be
well tolerated by the binding site. On the other hand, derivatives 4 and 5 were designed
to investigate the opposite end of the molecule, by replacing the piperidine ring with a
tetrahydroquinoline (4) or a tetrahydroisoquinoline (5).
Following the idea of Lounsbury and co-workers, to assess the relevance of the 2-
furanyl moiety of RNPA2000 [23], we also decided to evaluate some alternatives to the

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furanyl ring. Compounds
3,5-dichloro ( ), 3-chloro (
same phenylic moiety of JC1
6
–
9
present phenyl rings, which are substituted with halogens:
8), and 3,5-dibromo (9). We decided to mimic the
JC2, to appraise whether its specific electronic and steric
6
7), 3-bromo (
/
combinations are required for a strong inhibition of RnpA. Moreover, the productivity
of the substitution of heterocyclic moieties with halogen-phenyl rings has been widely
demonstrated [25]. The derivative 10 presents the 4-biphenyl-moiety in lieu of the 4-i-
propylphenyl one, as in compounds 1–3. Finally, compounds 11–14 are characterized
by differences in the linker between the two aromatic moieties. They were designed
to evaluate the importance of the semi thiocarbazide moiety of RNPA2000 in terms of
electronic density, linearity, polarity, and conformational freedom.
2. Results and Discussion
2.1. Computational Studies and Design
Using in-silico predictions, we aimed to estimate which cavity of the protein could
be responsible for the binding of JC-like compounds, RNPA2000, the set of derivatives
synthesized in this work and to decipher the main key interactions of these compounds in
the binding site.
With this objective and using S. aureus RnpA protein, a detailed Hotspots Maps calcu-
lation was performed. These results revealed that RnpA displays low druggability when
compared with other proteins, and only one low hotspot scoring area was identified as the
main ligandable binding site. Both JC1 and RNPA2000 were docked in this hypothetical
binding pocket in order to validate our hypothesis (Figure 3).
Figure 3. Hotspot maps calculated for the protein. Overlay between the hotspot maps and the
predicted binding mode for RNPA2000 and JC1 in the hypothesized binding site.
First, and given the unknown mechanism of action of RnpA inhibitors in RNA degra-
dation and ptRNA maturation, a blind docking study was carried out. For this study, we
considered the RnpA structure, and two reference compounds that were proposed in the
literature as RnpA inhibitors (JC1 and RNPA2000) described above to assess their predicted
binding cavity. The docking studies suggest that the reference molecules might be able
to bind RnpA in the main area that was identified previously in the hotspots maps. The
top part of this region involves the sequence of highest phylogenetic conservation in the
RnpA proteins being named the RNR box motif and represented by residues 59–67. This
positive-charged motif, containing three arginines and two lysines, resides at the top half of
α2 being solvent exposed, being poised to form the required interactions with the catalytic
RNA ribozyme (P-RNA) component of the complex [26].
Specifically, there are several key subareas in the solvent-exposed pocket that might
drive the ligand-protein interaction:

Antibiotics 2021, 10, 438
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•
•
•
in the subregion where the main hydrophobic hotspot was identified, there are several
aromatic and hydrophobic residues, including Tyr7, Phe43, Leu45, and Phe70, where
the first two are generating a cleft. This might help in anchoring aromatic systems,
establishing
π or CH–π interactions (such as the i-propylphenyl group that is present
in RnpA2000);
the central part contains a solvent-exposed area, which might allow the binding of
linear linkers by establishing hydrogen bonds interactions via the backbone of Ile9,
Leu45, Ile47 or the guanidinic moiety of Arg67. H-bonds donor-acceptor that are
predicted to interact with the ureidic portion of JC1 and with the semi-thiocarbazide
moiety of RNPA2000; and,
finally, at the other end, Phe15 and Lys63 are suitable for
interactions with different aromatic moieties, such as the one present in RNA2000.
According to the docking results, compound is predicted to bind the same cav-
π interaction or π-cation
1
ity achieving interactions that are close to the reference compounds mentioned above
(Figure 4a). The biphenyl motif engages with the aromatic residues on the cleft, while the
amide makes H-bond with Leu45, and Arg67, as shown in Figure 4a. On the other hand,
2
and 3 with a different substitution pattern in the biphenyl system and with the vector
growing at the 2 and 3 positions towards the solvent exposed area seem to be less attractive
to fit in the cleft region as compared to 1.
Figure 4. Predicted binding poses, based on docking studies, for different compounds designed in
this work: (
scaffold being the amide able to interact via H-bond with Leu45 and Arg67. (
where the carbonyl interacts with Arg67 and thiosemicarbazide makes H-bond with Ile9 and Leu45.
) Compound 10 (blue) making H-bond interaction with Ile9 and Ile47, and Arg67. ( ) Compound
11 (orange) making H-bond interaction with Leu45, Ile47, and Arg67 with the double amide moiety.
a) Compound
1
(light grey) docked in the putative binding site containing a biphenyl
b
) Compound (yellow)
6
(c
d

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Compounds
4
and
5
were designed to potentially achieve an extra-interaction with
the Phe15 or Lys63 at the binding site, by the substitution of the piperidine ring of JC1
with a tetrahydroquinoline or a tetrahydroisoquinoline, respectively. Compounds
6–9
showed a very good interaction profile and fit in the pocket, with the i-propylphenyl
and the semi-thiocarbazide showing a very similar shape to RNPA2000 and an optimal
linearity given by the linker. At the other end, the replacement of the furanyl moiety by
different halogen-phenyl systems are tolerated, as depicted in Figure 4b with compound
6. While considering derivative 10, the biphenylic portion was introduced to replace the
characteristic i-propylic substituent present in other RNPA2000-like structures and evaluate
its effects. Compound 10 keeps the H-bond interactions between the linker and Ile9, Ile47
and Arg67 and the biphenyl achieves a perfect fit in the cleft (Figure 4c).
Finally, compounds 11–14 were designed to evaluate the importance of the semi-
thiocarbazide moiety. Even removing the semi-thiocarbazide and replacing it with a
di-amidic structure, these compounds maintain some of the key interactions with RnpA, as
shown in Figure 4d. Moreover, this series of compounds should be more metabolically sta-
ble than RNPA2000-like derivatives, which might contain a liable nitrogen-nitrogen bond.
From a computational point of view, the hotspot maps analysis, together with the
docking studies, suggested the binding cavity for this set of compounds proposing their
putative binding mode and their main interactions with RnpA. In addition, several struc-
tural modifications were proposed to test different hypothesis and evaluate the effect of
these modifications on both the phenotypic assay and the capability of these compounds to
inhibit in-vitro RnpA-mediated ptRNA maturation. These results will need further struc-
tural confirmation in future studies, given the lack of structural information for inhibitors
of this route.
2.2. Chemistry
Scheme 1 shows the synthesis of compounds 1 to 5. The synthetic route is the same
for each compound. The proper carboxylic acid was converted into the corresponding
isocyanate by treatment with diphenyl phosphoryl azide and subsequent Curtius rear-
rangement after heating, and soon reacted with the appropriate amine, yielding the desired
ureidic derivative.
Scheme 1. Reagents and solvents: (
a
) Diphenyl phosphoryl azide (DPPA), Triethylamine (TEA), toluene,
◦
◦
◦
95 C, 40 min; (
b
) 2-methylpiperidine, toluene, 95 C; (
c) 1,2,3,4-tetrahydroquinoline, toluene, 95 C;
◦
and, (d) 1,2,3,4-tetrahydroisoquinoline, toluene, 95 C.
For the obtainment of the final compounds 6 to 9 (Scheme 2), we followed the synthetic
route that was described by Lounsbury and colleagues [23], in which the key intermediate
2-(i-propylphenoxy)acetohydrazide is treated with the proper acyl isothiocyanate to obtain
the thiosemicarbazide group, peculiar for this four compounds. To do so, we achieved the
key intermediate 16 starting from the commercially available 4-i-propylphenol, which was

Antibiotics 2021, 10, 438
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alkylated by reaction with methyl 2-chloroacetate in the presence of K2CO3 as base and
subsequently transformed into the corresponding hydrazide by treatment with hydrazine
hydrate in methanol.
◦
Scheme 2. Reagents and solvents: (
hydrate, Methanol, Reflux, 16 h; (
temperature (RT), 1 h; and, (e) Acetonitrile, RT, 30 min.
a
c
) Methyl 2-chloroacetate, K2CO3, DMF, 50 C, 1.5 h; (
b
) Hydrazine
) SOCl2, Reflux, 1 h; (d) Potassium thiocyanate, Acetonitrile, room
The synthetic scheme that was performed for the synthesis of compound 10
(Scheme 3)
follows the one just described for compounds to . In this case, the 4-phenylphenol
6
9
was converted into the 2-(4-biphenyloxy)acetohydrazide, tracing the same strategy de-
scribed before, and treated with the 3,5-dichlorobenzoyl isothiocyanate, yielding the desired
compound 10.
◦
Scheme 3. Reagents and solvents: (
a
) Methyl 2-chloroacetate, K2CO3, DMF, 50 C, 1.5 h; (
b) Hydrazine
hydrate, methanol, reflux, 16 h; (c) SOCl2, reflux, 1 h; (d) Potassium thiocyanate, acetonitrile, RT, 1 h;
and, (e) Acetonitrile, RT, 30 min.
The synthesis of compounds 11–12 (Scheme 4), characterized by two amidic groups
linked at the nitrogen atoms by methylenic or ethylenic linker (compounds 11 and 12), is
based on the same strategy. Firstly, the two moieties were reacted with the appropriate
monoprotected amine, generating the first amidic group. Thus, the intermediate was depro-
tected and linked to the second moiety to achieve the second amidic group. In particular,
for compound 11, the process started from intermediate 15, which was hydrolyzed and
treated with the N-Boc-methylendiamine, being adequately prepared, as described by
DeBons and Loudon [27]. Therefore, this intermediate was deprotected and converted
into the desired compound 11. For compound 12, conversely, the synthesis started from
the 3,5-dichlorobenzoic acid, which undergoes the same reactions as before, using the
appropriate amine.

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Scheme 4. Reagents and solvents: (
amine, TEA, DCM, RT, 3 h; ( ) 10% aqueous HCl, MeOH, reflux, 0.5 h; (
h; (f) N-Boc ethylenediamine, TEA, DCM, RT 3 h, ad (e) Intermediate 20, TEA, DCM, RT, 1 h.
a) 2.5 N aqueous NaOH, MeOH, RT, 1.5 h; (
b) SOCl2, reflux, 1 h; (c) N-Boc methylenedi-
d
e) 3,5-dichlorobenzoyl chloride, TEA, DCM, RT, 1.5
Lastly, the obtainment of compounds 13–14 (Scheme 5) was possible by following the
same strategy previously described, since also these two derivatives are characterized by a
diamidic structure. In this case, the intermediate 20 was treated with the corresponding
amine, 4-N-Boc-aminopiperidine and N-Boc-piperazine, to achieve the first amidic group
( –26). Therefore, these intermediates were deprotected (27–28) and treated with 3,5-
25
dichlorobenzoyl chloride yielding the final derivatives.
Scheme 5. Reagents and solvents: (a) 4-N-Boc-aminopiperidine or N-Boc-piperazine, TEA, DCM, RT, 3 h; (b) TFA, DCM, RT,
18 h; (c) 3,5-dichlorobenzoyl chloride, TEA, DCM, RT, 2 h.
2.3. Biological Evaluation
2.3.1. Antimicrobial Activity
The antimicrobial activities of compounds
1–14 (Table 1) were evaluated on two
S. aureus strains: a methicillin-sensitive S. aureus (MSSA, ATCC 29213) and a methicillin-
resistant S. aureus (MRSA, ATCC 43300). The inhibitory ability of compounds 1–14 was
evaluated by determining the minimum inhibitory concentration (MIC).
Some of the tested compounds displayed very promising antimicrobial activities, with
reasonable MICs ( , and 10), as shown in Table 1. Next, we assessed whether these
antimicrobial activities correlated with the inhibition of RnpA activities.
6, 7, 8, 9

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Table 1. Antimicrobial activity of compounds 1–14.
Compound Name
MSSA (ATCC 29213)
MRSA (ATCC 43300)
MIC (µM)
MIC (µM)
1
2
3
>500
>500
>500
311
>500
>500
>500
311
4
5
6
>500
21.1
>500
21.1
7
8
24.7
22.2
24.7
22.2
9
>500
21.1
>500
>500
>500
>500
18.9
21.1
>500
>500
>500
>500
10
11
12
13
14
2.3.2. In-Vitro Assays
Firstly, we evaluated the capability of these compounds to inhibit in-vitro RnpA-
mediated ptRNA maturation. To do so, Rnase P (RnpA + rnpB) was reconstituted and
combined with the putative inhibitor or DMSO (as a negative control) and ptRNATyr for
30 min, and the relative quantities of ptRNATyr and mature tRNA were evaluated by Urea-
PAGE. Figure 5 depicts representative data of the performance of compounds
presence of RNase P alone (Figure 5, lane 3) catalyzes the conversion of ptRNA into mature
tRNA. At the same time, increasing amounts of compounds and partially perturb the
6 and 7. The
6
7
activity of RNase P, resulting in the accumulation of ptRNA (Figure 5, lanes 4 to 9). All of
the compounds were tested at least twice and Table 2 records their IC50 values.
In parallel, the mRNA degradation inhibition profile of each compound was also
evaluated. The time course experiments were performed by incubating 62.5 µM of the
compound (or DMSO) with RnpA in the presence of a fluorescently labeled RNA substrate
for 30 min. RnpA efficiently degraded RNA in the presence of DMSO (circles), but it
was inhibited to 40–60% in the presence of the compounds 6 and 7, as shown in Figure 6
(triangles and squares, respectively). Subsequent dose response assays were carried out
using increasing concentrations of each analogue to determine the IC50 value of each
compound (Table 2).
Figure 5. In-vitro ptRNA maturation assay for Compounds 6 and 7. 0, 62.5, 250, or 500 µM compound
◦
was incubated with 10 pmol RNase P and ptRNATyr for 30 min at 37 C and conversion of ptRNA to
tRNA was evaluated by urea-PAGE; migration of tRNATyr and ptRNATyr are shown.

Antibiotics 2021, 10, 438
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Table 2. IC50 values of compounds 1–14.
Compound Name
RNPA2000
Degradation IC50
275
*
Processing IC50 **
140
1
2
3
72.5
233
324
66
36
37
>500
50
4
5
6
>500
53
75
59
7
8
77
49
28
76
9
-
-
33
153
423
>500
25
10
11
12
13
14
188
31
165
198
174
* Degradation IC50 = in vitro mRNA degradation IC50; ** Processing IC50 = in vitro ptRNA processing IC50
.
5000
DMSO
4000
Cpd 6
3000
Cpd 7
2000
1000
0
10
20
30
40
−
-1000
Minutes
Figure 6. In-vitro mRNA degradation assay—Compounds
6
and
7. Representative analogue in-
hibition of in-vitro RnpA-mediated RNA degradation. DMSO or 62.5
incubated with RnpA in the presence of the FRET RNA substrate for 30 min at 37 C. Fluorescence
measurements (RFUs) were collected every 2 min at excitation 490 nm, emission 520 nm.
µ
M of the◦compound was
Table 2 summarizes the IC50 values for each compound, which were calculated for
both mRNA degradation inhibition and ptRNA maturation inhibition. The results revealed
that the substitution of the 3,5-dichlorophenyl moiety of JC1
always detrimental for the inhibition of the RnpA-mediated processes and, consequently,
for the antimicrobial activity, although the 4-biphenilic derivative presents a better overall
/JC2 with a biphenylic one is
1
profile when compared to the 3-biphenilic and the 2-biphenilic derivatives 2 and 3. These
data match with the in-silico prediction. Conversely, the tetrahydroquinolinic compound
4
shows stronger RnpA inhibition properties as compared to the tetrahydroisoquilinic
derivative . Despite that these results seem to be in contrast with the predicted docking
data, the different activity might be due to other factors rather that the inability to correctly
interact with RnpA. The IC50 values of compounds , and, partially, confirm the
productivity of the substitution of the 2-furanylmoiety with halogen-phenyl rings. In
particular, the dichlorophenyl derivative present comparative inhibition profiles for
mRNA degradation and ptRNA processing, while derivatives (m-chloro) and (m-
bromo) show, respectively, better ptRNA processing inhibition and mRNA degradation
inhibition capability. Compounds 11 14 present no antimicrobial activity, which suggests
the importance of the semi-thiocarbazide moiety.
5
6,
7
,
8
9
6
7
8
–

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2.3.3. Cellular Assays
S. aureus were treated with 0.5
×
or 1
×
MIC of each compound for 1 h and qRT-PCR
was used to quantify the accumulation of ptRNAtyr in order to evaluate the capability of
each compound to affect RnpA-mediated ptRNA processing in the more complex cellular
system, as previously described [22
accumulate ptRNATyr, whereas RNPA2000 (positive control) resulted in a dose-dependent
accumulation of 2.5-fold and 3.8-fold at 0.5 and 1 MIC, respectively, as shown in
Figure 7. Compound
confirmed its ability to inhibit RnpA-mediated ptRNATyr processing
and also demonstrated a dose-dependent effect on ptRNATyr accumulation. Treatment
with compound , which had inhibitory activity in vitro, resulting in a 2.23-fold increase
in ptRNATyr accumulation at 0.5 MIC, but did not inhibit ptRNATyr processing after
treatment with 1 MIC, possibly due to the compound precipitation at this concentration.
Compound 10, which had a good in-vitro inhibition of ptRNATyr processing, resulted
in 1.4-fold and 1.3-fold increases in ptRNATyr accumulation at 0.5
and 1 of the MIC,
,24]. Cells that were treated with DMSO did not
×
×
7
8
×
×
×
×
respectively. Treatment with compounds
4 and 6 did not result in the accumulation of
ptRNATyr, despite the antistaphylococcal and inhibitory activity in vitro.
Figure 7. ptRNA accumulation cellular assay. Quantitative RT-PCR measures of ptRNATyr accumu-
lation in S. aureus UAMS-1 cells treated with DMSO, analogues or RNPA2000 (positive control). Cells
were treated with 0.5
were averaged.
×
or 1
×
MIC for 1 h. Each compound was evaluated twice, and the values
As a more direct measure of the effect of each analogue, on cellular mRNA degradation,
RnpA-mediated turnover of spa RNA, a natural substrate of RnpA, was evaluated. S. aureus
UAMS-1 was treated with 0.5
(PTA) with rifampin and spa RNA was quantified via RT-PCR at 0- and 5-min PTA, as
previously described [ ]. In cells that were treated with DMSO, 55% of spa transcript
was degraded after 5 min PTA, as shown in Figure 8. The RNPA2000 positive control, as
well as compounds and , showed a significantly reduced degradation of spa transcript.
Compound had no effect on cellular RNA turnover, with the treated cells degrading
approximately 66% of spa after 5 min PTA, despite having in vitro activity. Compound
which also had in vitro activity, resulting in an average of 31% spa transcript degradation,
an approximate 24% inhibition of degradation when compared to DMSO. Compound 10
×
the MIC for 30 min prior to post-transcriptional arrest
9
7
8
4
6
,
,
which had weak in vitro degradation activity, had an average inhibition of 22% on cellular
RNA turnover in comparison to DMSO.

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Figure 8. RT-PCR of spa transcript turnover. S. aureus UAMS-1 cells were treated with 0.5
of each compound for 30 min prior to transcriptional arrest (PTA). Each compound was evaluated
twice, and values were averaged.
×
MIC
Taken together, compounds
7 and 8, presenting a 3-halogenphenyl moiety in place of
the 2-furanyl one, performed well when evaluated in in-vitro assays and they were the top
performers in both cellular assays, significantly inhibiting both RnpA-mediated cellular
RNA metabolism functions. Although compound 10, in which the i-propylphenyloxy
portion was replaced by a more lypophilic one, had the most potent antibacterial activity,
it had weak effects on RNA degradation (both in vitro and in vivo), but it was a good
inhibitor of ptRNATyr processing in vitro and it had significantly increased ptRNATyr
accumulation in cellular assays.
3. Conclusions
In the present work, we predicted key interactions that RNPA2000 and JC1/2 achieve
with their molecular target RnpA. This information guided the design of new inhibitors,
which increased the knowledge of the SAR of RNPA2000-like and JC1/2-like RnpA in-
hibitors. The lower activity of compounds
1 to 5 as compared to JC1/2 suggest that the
combination of a small-aliphatic amine with a 3,5-dichlorophenyl moiety is required for
RnpA inhibition. Nevertheless, further investigation regarding this class of compounds
remains worthwhile. Compounds
of the 2-furanyl moiety, demonstrated cellular activity that was comparable to RNPA2000
while avoiding the potentially metabotoxic furan. At the same time, the inactivity of
compounds 11 12 13, and 14 underline the importance of the semi-thiocarbazide. This
7 and 8, characterized by a 3-halogenphenyl ring in place
,
,
,
preliminary set of data, which are perfectly in line with the innovative computational
model, trace the path for a deeper investigation of the SAR of these very promising classes
of compounds. Moreover, an exciting aspect of the current work is the development
of a putative computational-based path for RnpA inhibitor discovery and optimization.
Building from our early modeling success is expected to allow for the rational design of
more potent and presumably more selective molecules that, as they become available and
evaluated for RnpA inhibitor properties, are expected to allow refining the computational
modeling in iterative manner and yielding higher quality probes for mutagenesis based
testing of the model.
4. Materials and Methods
4.1. Chemistry
All of the reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and
they were used without further purification. The solvents, such as ACN, THF, DCM, DMF,
methanol, and acetone, were purchased from Sigma Aldrich.
Silica gel matrix, with fluorescent indicator 254 nm, was used in analytical thin-layer
chromatography (TLC on aluminum foils), and silica gel (particle size 40–63 µm, Merck)
was used in flash chromatography on Sepachrom Puriflash XS 420. Visualizations were
accomplished with UV light (λ 254 or 280 nm).

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The 1H-NMR spectra were measured by Varian Mercury 300 NMR spectrometer/Oxford
Narrow Bore superconducting magnet operating at 300 MHz. The 13C-NMR spectra were
acquired operating at 75 MHz. Chemical shifts (δ) are reported in ppm relative to residual
solvent as the internal standard. Signal multiplicity is used according to the following
abbreviations: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet,
dq = doublet of quadruplets, m = multiplet, bs = broad singlet, and set = septuplet. The
final products,
column (5 m, 4.6 mm
detector, with different methods (Table 3). Their purity was quantified at peculiar
1
–
14, were analyzed by reverse-phase HPLC using a Waters XBridge C-18
150 mm) on an Elite LaChrom HPLC system with a diode array
max,
µ
×
λ
depending on each sample, and it resulted to be >95%. The relative retention times are re-
ported in each experimental section. The melting points were determined by DSC analysis
over a TA Instruments DSC 1020 apparatus. The 1H- and 13C-NMR spectra of compounds
1–14, together with their HPLC profiles, are included in the supplementary material S1.
Table 3. HPLC Methods for the analysis of compounds 1–14.
Method Name
Mobile Phase
Flow Rate (mL/min)
A
B
C
D
H2O (1‰ TFA)/ACN (1‰ TFA) 55/45
H2O (1‰ TFA)/ACN (1‰ TFA) 55/45
H2O (1‰ TFA)/ACN (1‰ TFA) 1/1
H2O (1‰ TFA)/ACN (1‰ TFA) 40/60
1
1.5
1.5
1.5
Synthesis
1-(4-Biphenyl)-3-(2-methylpiperdin-1-yl)urea (
1): diphenyl phosphoryl azide (DPPA)
(2.71 mL, 12.10 mmol) was added dropwise to a solution of biphenyl-4-carboxylic acid
(2.00 g, 10.09 mmol) and triethylamine (TEA) (1.69 mL, 12.10 mmol) in toluene (20 mL).
The reaction mixture was stirred at RT for 20 min, heated at 95 ◦C, and then stirred again
for 20 min. The mixture was cooled at RT, added with 2-methylpiperidine (1.25 mL,
10.59 mmol), heated again at 95 ◦C and stirred overnight. Then, the mixture was diluted
with ethyl acetate (30 mL), washed twice with 10% aqueous NaHCO3, dried over Na2SO4,
filtered, and concentrated under vacuum to give a residue, which was purified by flash
chromatography. Elution with cyclohexane/ethyl acetate 80/20 and subsequent crystal-
lization from ethanol (15 vol) gave 0.85 g of 1
as a white solid. Yield: 29% M.p: 164.21 ◦C.
Tr (HPLC): 12.65 min. (Method A), Purity = 99.7%. 1H NMR (300 MHz, CDCl3, δ) 7.55
(m, 4H), 7.46 (m, 4H), 7.29 (m, 1H), 6.49 (bs, 1H), 4.39 (m, 1H), 3.91 (d, J = 12.8 Hz, 1H),
3.02 (t, J = 12.8 Hz, 1H), 1.62 (m, 6H), 1.25 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3,
δ) 154.94, 140.76, 138.79, 135.57, 128.68, 127.41, 126.77, 126.71, 120.13, 46.74, 39.13, 30.23,
25.63, 18.55, 15.76 ppm.
1-(3-Biphenyl)-3-(2-methylpiperdin-1-yl)urea (
2): Prepared from biphenyl-3-carboxylic
acid as described for using DPPA (1.2 eq), TEA (1.2 eq), and 2-methylpiperdine (1.05 eq)
1
in toluene (10 vol), and then purified by flash chromatography on silica gel. Elution with
cyclohexane/ethyl acetate 80/20 and subsequent crystallization from EtOH (4 vol) a◦nd
recrystallization from IPA (4 vol) gave 0.52 g of 2 as a white solid. Yield: 17% M.p: 126.53 C.
Tr (HPLC): 12.67 min. (Method A), Purity = 99.3%. 1H NMR (300 MHz, CDCl3, δ) 7.65 (m,
3H), 7.32 (m, 6H), 6.46 (bs, 1H), 4.41 (m, 1H), 3.91 (d, J = 13.0 Hz, 1H), 3.02 (t, J = 13.0 Hz,
1H), 1.64 (m, 6H), 1.25 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3, δ) 154.94, 141.91,
140.98, 139.75, 129.16, 128.61, 127.26, 127.18, 121.64, 118.72, 118.68, 46.76, 39.13, 30.23, 25.63,
18.55, 15.77 ppm.
1-(2-Biphenyl)-3-(2-methylpiperdin-1-yl)urea (
3): Prepared from biphenyl-2-carboxylic
acid as described for using DPPA (1.2 eq), TEA (1.2 eq). and 2-methylpiperdine (1.05 eq)
1
in toluene (10 vol), and the purified by flash chromatography on silica gel. Elution with
cyclohexane/ethyl acetate 80/20 and subsequent crystallization from IPA (4 vol) gave
1.02 g of 3
as a white solid. Yield: 35% M.p: 104.05 ◦C. Tr (HPLC): 10.67 min. (Method A),
Purity = 96.4%. 1H NMR (300 MHz, CDCl3, δ) δ 8.11 (d, J = 8.1 Hz, 1H), 7.41 (m, 6H), 7.20
(dd, J = 7.6, 1.6 Hz, 1H), 7.07 (td, J = 7.6, 0.9 Hz, 1H), 6.47 (bs, 1H), 3.98 (m, 1H), 3.70 (d,

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J = 12.6 Hz, 1H), 2.78 (t, J = 12.6 Hz, 1H), 1.44 (m, 6H), 1.01 (d, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3, δ) 154.68, 138.72, 136.43, 131.52, 129.52, 129.32, 128.98, 128.38, 127.77,
122.49, 120.85, 46.62, 38.57, 30.19, 25.46, 18.52, 15.58 ppm.
1-(3,5-Dichlorophenyl)-3-(1,2,3,4-tetrahydroquinolin-1-yl)urea (
4): Prepared from 3,5-
dichlorobenzoic acid, as described for using DPPA (1.2 eq), TEA (1.2 eq), and 1,2,3,4-
1
tetrahydroquinoline (1.05 eq) in toluene (10 vol) and purified by flash chromatography on
silica gel. Elution with cyclohexane/ethyl acetate 80/20 and subsequent crystallization
from EtOH (6 vol) gave 0.80 g of
4
as a white solid. Yield: 32% M.p: 143.34 ◦C. Tr (HPLC):
12.46 min. (Method C), Purity = 99.8%. 1H NMR (300 MHz, CDCl3, δ) 7.34 (d, J = 1.9 Hz,
2H), 7.25 (m, 3H), 7.15 (m, 1H), 7.06 (bs, 1H), 7.00 (t, J = 1.8 Hz, 1H), 3.81 (t, J = 6.3 Hz, 2H),
2.79 (t, J = 6.7 Hz, 2H), 1.98 (m, 2H). 13C NMR (75 MHz, CDCl3, δ) 152.98, 140.59, 138.25,
135.11, 133.21, 129.89, 127.05, 125.32, 122.88, 122.82, 117.15, 43.43, 26.90, 23.94 ppm.
1-(3,5-Dichlorophenyl)-3-(1,2,3,4-tetrahydroisoquinolin-1-yl)urea (
5): Prepared from 3,5-
dichlorobenzoic acid as described for using DPPA (1.2 eq), TEA (1.2 eq), and 1,2,3,4-
1
tetrahydroisoquinoline (1.05 eq) in toluene (10 vol), and then purified by flash chromatog-
raphy on silica gel. Elution with cyclohexane/ethyl acetate 85/15 and subsequent crys-
tallization from IPE (20 vol) gave 0.60 g of
5
as a white solid. Yield: 24% M.p: 128.79 ◦C.
Tr (HPLC): 9.00 min. (Method C). Purity = 99.9%. 1H NMR (300 MHz, CDCl3, δ) 7.36 (d,
J = 1.5 Hz, 2H), 7.18 (m, 4H), 6.98 (s, 1H), 6.75 (bs, 1H), 4.64 (s, 2H), 3.71 (t, J = 5.8 Hz, 2H),
2.92 (t, J = 5.7 Hz, 2H). 13C NMR (75 MHz, CDCl3, δ) 154.29, 141.04, 134.92, 134.68, 132.73,
128.39, 126.99, 126.61, 126.29, 122.85, 118.15, 45.77, 41.73, 28.89 ppm.
Methyl 4-i-propylphenoxyacetate (15): methyl chloroacetate (0.71 mL, 8.01 mmol) was
added to a solution of 4-isopropylphenol (1.00 g, 7.34 mmol) and potassium carbon-
◦
ate (1.12 g, 8.01 mmol) in DMF (10 mL). The reaction mixture was stirred at 50 C for
1.5 h, concentrated under vacuum, diluted with ethyl acetate (30 mL), washed with brine
(
4 × 10 mL), dried over Na2SO4, filtered, and then concentrated under vacuum to give
1.4 g of 15 as a yellowish oil. Yield: 92%. 1H NMR (300 MHz, CDCl3, δ) 7.14 (d, J = 8.7 Hz,
2H), 6.84 (d, J = 8.7 Hz, 2H), 4.61 (s, 2H), 3.80 (s, 3H), 2.86 (set, J = 6.9 Hz, 1H), 1.22 (d,
J = 6.9 Hz, 6H).
4-i-Propylphenoxyacetohydrazide (16): hydrazine hydrate (2.1 mL, 33.60 mmol) was
added to a solution of 15 (1.40 g, 6.72 mmol) in methanol (15 mL). The reaction mixture was
stirred at reflux for 16 h, concentrated under vacuum, diluted with ethyl acetate (30 mL),
washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum
◦
to give 1,36 g of 16 as a white solid. Yield = 97%. M.p: 105.21 C. 1H NMR (300 MHz,
CDCl3, δ): 7.16 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 8.2 Hz, 2H), 4.55 (s, 2H), 2.87 (set, J = 6.9 Hz
1H), 1.22 (d, J = 6.9 Hz, 6H).
,
1-(4-i-Propylphenoxyacetyl)-4-(3,5-dichlorobenzoyl)thiosemicarbazide (6): a solution of 3,5-
dichlorobenzoic acid (1.30 g, 6.8 mmol) in SOCl2 (6.5 mL) was stirred at reflux for 1 h.
Thus, the solution was concentrated under vacuum, diluted with acetonitrile (15 mL),
and then added with potassium thiocyanate (1.12 g, 11.56 mmol). The reaction mixture
was stirred at RT for 1 h, added with a solution of 16 (1.40 g, 6.8 mmol) in DMF (5 mL),
stirred at RT for 30 min, concentrated under vacuum, diluted with ethyl acetate (50 mL),
washed with brine (4
vacuum to give a solid. Digestion firstly with MeOH (15 vol) at reflux and secondly with
ACN (15 vol) at reflux gave 1.3 g of
as a yellowish solid. Yield: 43%. M.p: 195.69 ◦C. Tr
×
10 mL), dried over Na2SO4, filtered, and then concentrated under
6
(HPLC): 17.60 min. (Method C), Purity = 99.6%. 1H NMR (300 MHz, DMSO-d6, δ) 12.38
(bs, 1H), 11.95 (bs, 1H), 11.00 (bs, 1H), 7.96 (d, J = 1.8 Hz, 2H), 7.89 (t, J = 1.8 Hz, 1H), 7.15
(d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 4.68 (s, 2H), 2.82 (set, J = 6.9 Hz, 1H), 1.16 (d,
J = 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, δ) 177.66, 165.66, 165.61, 156.23, 141.70,
135.56, 134.63, 132.58, 127.88, 127.55, 115.02, 66.24, 33.06, 24.50 ppm.
1-(4-i-Propylphenoxyacetyl)-4-(3-chlorobenzoyl)thiosemicarbazide (
chlorobenzoic acid, as described for using SOCl2 (5 vol), potassium thiocyanate (1.6 eq)
and 16 (1 eq) in acetonitrile (10 vol) at RT for 30 min. Digestion of the crude product with
IPA (10 vol) at reflux gave 1.06 g of
as a pale yellow solid. Yield: 27%. M.p: 187.27 ◦C. Tr
7): Prepared from 3-
6
7

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(HPLC): 9.38 min. (Method C), Purity = 98.5%. 1H NMR (300 MHz, DMSO-d6, δ) 12.47
(bs, 1H), 11.90 (bs, 1H), 11.01 (bs, 1H), 8.00 (t, J = 1.9 Hz, 1H), 7.86 (m, 1H), 7.70 (ddd, J = 8.0,
1.9, 1.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 4.68
(s, 2H), 2.82 (set, J = 6.9 Hz, 1H), 1.15 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, δ)
178.09, 167.00, 156.66, 156.24, 141.69, 134.31, 133.56, 133.25, 130.81, 128.92, 127.91, 127.59,
115.00, 66.16, 33.05, 24.54 ppm.
1-(4-i-Propylphenoxyacetyl)-4-(3-bromobenzoyl)thiosemicarbazide (
8): Prepared from 3-
bromobenzoic acid as described for using SOCl2 (5 vol), potassium thiocyanate (1.6 eq)
6
and 16 (1 eq) in acetonitrile (10 vol) at RT for 30 min. Digestion of the crude product with
◦
IPA (5 vol) at reflux gave 1.96 g of
8
as a white solid. Yield: 62%. M.p: 184.01 C. Tr (HPLC):
10.13 min. (Method C), Purity = 98.5%. 1H NMR (300 MHz, DMSO-d6, δ) 12.46 (bs, 1H),
11.90 (bs, 1H), 11.03 (bs, 1H), 8.14 (t, J = 1.7 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.83 (m, 1H),
7.47 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 4.68 (s, 2H), 2.81 (set,
J = 6.9 Hz, 1H), 1.15 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, δ) 178.13, 166.89,
165.67, 156.24, 141.69, 136.13, 134.50, 131.73, 131.04, 128.28, 127.59, 121.92, 114.99, 66.15,
33.05, 24.54 ppm.
1-(4-i-Propylphenoxyacetyl)-4-(3,5-dibromobenzoyl)thiosemicarbazide (
3,5-dibromobenzoic acid, as described for using SOCl2 (5 vol), potassium thiocyanate
(1.6 eq), and 16 (1 eq) in acetonitrile (10 vol) at RT for 30 min. Digestion of the crude product
firstly with MeOH (10 vol) and secondly with IPA (15 vol) at reflux gave 2.00 g of as a
9): prepared from
6
9
white solid. Yield: 62%. M.p: 201.83 ◦C. Tr (HPLC): 6.19 min. (Method D), Purity = 99.0%.
1H NMR (300 MHz, DMSO-d6, δ) 12.36 (bs, 1H), 11.97 (bs, 1H), 11.03 (bs, 1H), 8.11 (s, 3H),
7.15 (d, J = 8.3 Hz, 2H), 6.87 (dd, J = 8.3 Hz, 2H), 4.67 (s, 2H), 2.82 (set, J = 6.9 Hz, 1H), 1.15
(d, J = 6.9 Hz, 7H). 13C NMR (75 MHz, DMSO-d6, δ) 177.89, 165.68, 165.48, 156.24, 141.68,
137.88, 136.01, 131.04, 131.04, 127.59, 122.81, 114.99, 66.14, 33.05, 24.54 ppm.
Methyl 4-biphenyloxyacetate (17): prepared from 4-hydoxybiphenyl as described for 15
using methyl 2-chloroacetate (1.25 eq) and potassium carbonate (1.2 eq) in DMF (10 vol) to
give 2.85 g of 17 as a white solid. Yield: 93%. M.p: 100.98 ◦C. 1H NMR (300 MHz, CDCl3,
δ) 7.53 (m, 4H), 7.41 (m, 2H), 7.31 (m, 1H), 6.99 (d, J = 8.8 Hz, 2H), 4.68 (s, 2H), 3.83 (s, 3H).
4-Biphenyloxyacetohydrazide (18): prepared from 17 as described for 16 using hydrazine
hydrate (5 eq) in MeOH (30 mL) to give 1,85 g of 18 as a white solid. Yield: 64%.
◦
M.p: 112.86 C. 1H NMR (300 MHz, DMSO-d6, δ): δ 9.43 (bs, 1H), 7.58 (m, 4H), 7.41
(t, J = 7.4 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.02 (dd, J = 9.3, 2.4 Hz, 2H), 4.53 (s, 2H), 4.33
(s, 2H).
1-(4-Biphenyloxyacetyl)-4-(3,5-dichlorobenzoyl)thiosemicarbazide (10): prepared from 3,5-
diclorobenzoic acid as described for 6 using SOCl2 (5 vol), potassium thiocyanate (1.6 eq)
and 18 (1 eq) in acetonitrile (10 vol) at RT for 1 h. Digestion of the crude product w◦ith
MeOH (10 vol) at reflux gave 2.77 g of 10 as a white solid. Yield: 73%. M.p: 200.71 C.
Tr (HPLC): 13.81 min. (Method C), Purity = 95.2%. 1H NMR (300 MHz, DMSO-d6, δ)
12.37 (bs, 1H), 11.98 (bs, 1H), 11.12 (bs, 1H), 7.96 (d, J = 1.9 Hz, 2H), 7.89 (t, J = 1.9 Hz, 1H),
7.59 (m, 4H), 7.42 (t, J = 7.6 Hz, 2H), 7.33–7.25 (m, 1H), 7.08 (d, J = 8.8 Hz, 2H), 4.77 (s,
2H). 13C NMR (75 MHz, DMSO-d6, δ) 178.03, 165.63, 165.56, 157.78, 140.12, 135.70, 134.58,
133.79, 132.60, 129.31, 128.20, 127.92, 127.27, 126.69, 115.64, 66.11 ppm.
4-i-Propylphenoxyacetic acid (19): 2.5 N aqueous NaOH (6 mL, 15 mmol) was added to
a solution of 15 (3.00 g, 14.40 mmol) in MeOH (30 mL). The reaction mixture was stirred
at RT for 1.5 h, concentrated under vacuum, diluted with water, acidified to pH = 1 with
conc. HCl, extracted twice with ethyl acetate (2
×
30 mL), dried over Na2SO4, filtered, and
◦
concentrated under vacuum to give 2.60 g of 19 as a white solid. Yield: 95%. M.p: 82.14 C.
1H NMR (300 MHz, CDCl3, δ) 7.16 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 4.66 (s, 2H),
2.86 (set, J = 6.9 Hz, 1H), 1.22 (d, J = 6.9 Hz, 6H).
4-i-Propylphenoxyacetyl chloride (20): SOCl2 (10 mL, 5 vol) was added to 19 (2.00 g,
10.30 mmol). The reaction mixture was stirred at reflux for 1 h and concentrated under
vacuum to give 2.15 g of 20 as a dark oil. Yield: 98%. 1H NMR (300 MHz, CDCl3, δ) 7.17

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(d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.92 (s, 2H), 2.87 (set, J = 6.9 Hz, 1H), 1.22 (d,
J = 6.9 Hz, 6H).
N-(t-Butoxycarbonylaminomethyl)-4-i-propylphenoxyacetamide (21): A solution of N-Boc
methylendiamine (1.20 g, 8.21 mmol) in DCM (5 mL) was added to a solution of 20 (1.6 g,
◦
8.21 mmol) and TEA (1.37 mL, 9.85 mmol) in DCM (5 mL) at 0 C. The reaction mixture was
stirred at RT for 3 h, diluted with DCM (20 mL), washed twice with 10% aqueous NaHCO3
(2
×
10 mL) and once with brine (10 mL), dried over Na2SO4, filtered and concentrated
under vacuum to give 2.17 g of 21 as a yellowish oil. 1H NMR (300 MHz, CDCl3, δ) 7.50
(bs, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 5.57 (bs, 1H), 4.62 (t, J = 6.4 Hz,
2H), 4.45 (s, 2H), 2.86 (set, J = 6.9 Hz, 1H), 1.43 (s, 9H), 1.22 (d, J = 6.9 Hz, 6H).
N-(Aminomethyl)-2-(4-i-propylphenoxy)acetamide (22): 10% aqueous HCl (2.5 mL,
7.5 mmol) was added to a solution of 21 (1.00 g, 3.10 mmol) in MeOH (10 mL) at RT.
The reaction mixture was stirred at reflux for 0.5 h, concentrated under vacuum, diluted
with Et2O (30 mL) and extracted twice with 10% aqueous HCl (2
×
15 mL). The aqueous
phase was thus basified to pH = 12 and extracted three times with ethyl acetate (3 × 25 mL),
dried over Na2SO4, filtered and concentrated under vacuum to give 0.40 g of 22 as a yel-
lowish oil. Yield: 58%. 1H NMR (300 MHz, CDCl3, δ) 7.26 (bs, 1H), 7.16 (d, J = 8.4 Hz,
1H), 6.84 (d, J = 8.4 Hz, 1H), 4.46 (s, J = 20.8 Hz, 2H), 4.30 (s, 2H), 2.86 (set, J = 6.9 Hz, 1H),
2.36 (bs, 2H), 1.24 (dd, J = 14.8, 7.0 Hz, 6H).
3,5-Dichloro-N-((2-(4-i-propylphenoxy)acetamido)methyl)benzamide (11): prepared from 22
as described for 21 using 3,5-dichlorobenzoyl chloride (1 eq), TEA (1.2 eq) in DCM (20 mL)
at RT for 1.5 h. Digestion of the crude product with IPA (10 vol) at reflux gave 0.85 g of 11 as
◦
a white solid. Yield: 70%. M.p: 189.88 C. Tr (HPLC): 9.94 min. (Method B), Purity = 97.0%.
1H NMR (300 MHz, DMSO-d6, δ) 9.30 (t, J = 5.4 Hz, 1H), 8.63 (t, J = 5.4 Hz, 1H), 7.88
(d, J = 1.7 Hz, 2H), 7.80 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.68 (t,
J = 5.4 Hz, 2H), 4.46 (s, 2H), 2.79 (set, J = 6.9 Hz, 1H), 1.12 (d, J = 6.9 Hz, 6H).13C NMR
(75 MHz, DMSO-d6, δ) 168.74, 164.30, 156.20, 141.50, 137.40, 134.73, 131.32, 127.51, 126.69,
114.91, 67.21, 44.63, 33.01, 24.49 ppm.
N-(t-Butoxycarbonylaminoethyl)-3,5-dichlorobenzamide (23):
prepared from 3,5-
dichlorobenzoyl chloride as described for 21 using N-boc ethylendiamine (1.5 eq) and
TEA (1.5 eq) in DCM (10 vol) to give 2.85 g of 23 as a pale yellow oil. Yield: 81%. 1H NMR
(300 MHz, CDCl3, δ) 7.71 (d, J = 1.7 Hz, 2H), 7.57 (bs, 1H), 7.45 (t, J = 1.7 Hz, 1H), 5.07 (bs,
1H), 3.52 (m, 2H), 3.40 (m, 2H), 1.44 (s, 9H).
N-(2-Aminoethyl)-3,5-dichlorobenzamide (24): prepared from 23 as described for 22 using
10% aqueous HCl (2 eq) in MeOH (10 vol) to give 1.61 g of 24 as a brownish oil. Yield: 80%.
1H NMR (300 MHz, CDCl3, δ) 7.64 (d, J = 1.6 Hz, 2H), 7.43 (t, J = 1.6 Hz, 1H), 7.14 (bs),
3.46 (dd, J = 10.9, 5.4 Hz, 2H), 2.93 (t, J = 5.7 Hz, 2H), 1.67 (bs, 2H).
3,5-Dichloro-N-((2-(4-i-propylphenoxy)acetamido)ethyl)benzamide (12): prepared from 24
as described for 21 using 20 (1 eq), TEA (1.2 eq) in DCM (20 mL) at RT for 1 h. Digestion of
the crude product with IPA (10 vol) at reflux gave 1.68 g of 12 as a white solid. Yield: 59%.
M.p: 181.73 ◦C. Tr (HPLC): 7.21 min. (Method C), Purity = 97.1%. 1H NMR (300 MHz,
DMSO-d6, δ) 8.70 (t, J = 4.6 Hz, 1H), 8.16 (t, J = 4.6 Hz, 1H), 7.82 (d, J = 1.7 Hz, 2H), 7.78
(t, J = 1.7 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.40 (s, 2H), 3.33 (m,
4H), 2.77 (sec, J = 6.9 Hz, 1H), 1.12 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, δ)
168.87, 164.17, 156.31, 141.52, 138.12, 134.65, 130.89, 127.41, 126.52, 115.01, 67.70, 38.51, 33.02,
24.44 ppm.
4-t-Butoxycarbonylamino-1-(4-i-propylphenoxyacetyl)piperidine (25): prepared from 20 as
described for 21 using 4-N-Boc-aminopiperidine (1 eq), TEA (1.2 eq) in DCM (20 mL) at
RT for 3 h. Crystallization of the crude product with MeOH (7.5 vol) gave 2.80 g of 25 as a
white solid. Yield: 60%. M.p: 160,53 ◦C 1H NMR (300 MHz, CDCl3, δ) 7.13 (d, J = 8.5 Hz,
2H), 6.85 (d, J = 8.5 Hz, 2H), 4.63 (s, 1H), 4.44 (m, 2H), 3.95 (d, J = 13.1 Hz, 1H), 3.66 (bs,
1H), 3.13 (t, J = 12.2 Hz, 1H), 2.83 (m, 1H), 2.82 (set, J = 6.9 Hz, 1H) 1.97 (t, J = 12.5 Hz, 2H),
1.43 (s, 9H), 1.20 (d, J = 6.9 Hz, 6H).

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4-Amino-1-(4-i-propylphenoxyacetyl)piperidine (27): TFA (5.66 mL, 73.80 mmol) was
◦
added to a solution of 25 (2.78 g, 7.38 mmol) in DCM (30 mL) at 0 C. The reaction mixture
was stirred at RT for 18 h, diluted with DCM, washed once with 10% aqueous NaOH
(15 mL), once with brine (15 mL), dried over Na2SO4, filtered, and concentrated under
vacuum to give 2.04 g of 27 as a yellowish oil. Yield: Quantitative. 1H NMR (300 MHz,
CDCl3, δ) 7.13 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 4.64 (s, 2H), 4.44 (d, J = 13.6 Hz,
1H), 3.96 (d, J = 13.6 Hz, 1H), 3.10 (t, J = 12.4 Hz, 1H), 2.85 (m, 2H), 2.82 (set, J = 6.9 Hz, 1H),
1.84 (m, 2H), 1.30 (m, 2H), 1.21 (d, J = 6.9 Hz, 6H).
4-(3,5-Dichlorobenzoylamino)-1-(4-i-propylphenoxyacetyl)piperidine (13): prepared from 27
as described for 21 using 3,5-dichlorobenzoyl chloride (1 eq), TEA (1.2 eq) in DCM (20 mL)
at RT for 3 h. Crystallization of the crude product◦with MeOH (5 vol) at reflux gave 0.81 g
of 13 as a white solid. Yield: 43%. M.p: 135.90 C. Tr (HPLC): 14.83 min. (Method B),
Purity = 98.4%. 1H NMR (300 MHz, DMSO-d6, δ) 8.53 (d, J = 7.5 Hz, 1H), 7.86 (m, 2H),
7.78 (m, 1H), 7.12 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 8.2 Hz, 2H), 4.79 (d, J = 14.0 Hz, 1H), 4.71
(d, J = 14.0 Hz, 1H), 4.28 (d, J = 13.1 Hz, 1H), 4.02 (m, 1H), 3.86 (d, J = 13.1 Hz, 1H), 3.15
(t, J = 12.1 Hz, 1H), 2.82 (set, J = 6,8 Hz, 1H), 2.80 (m, 1H), 1.85 (m, 2H), 1.53 (m, 1H), 1.37
(m, 1H), 1.15 (d, J = 6.8 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, δ) 166.23, 163.25, 156.59,
141.21, 138.11, 134.66, 131.00, 127.47, 126.59, 114.79, 66.69, 47.27, 43.69, 33.03, 32.16, 31.40,
24.55 ppm.
4-t-Butoxycarbonyl-1-(4-i-propylphenoxyacetyl)piperazine (26): Prepared from 20 as de-
scribed for 21 using N-Boc-piperazine (1 eq), TEA (1.2 eq) in DCM (20 mL) at RT for 3 h to
give 1.86 g of 26 as a yellow oil. Yield: Quantitative. 1H NMR (300 MHz, CDCl3, δ) 7.14
(d, J = 7.1 Hz, 2H), 6.86 (d, J = 7.1 Hz, 2H), 4.66 (s, 2H), 3.57 (m, 4H), 3.43 (m, 4H), 2.85 (set,
J = 6.8 Hz, 1H), 1.45 (s, 9H), 1.21 (d, J = 6.8 Hz, 6H).
1-(4-i-Propylphenoxyacetyl)piperazine (28): prepared from 26 as described for 27 using
TFA (10 eq) in DCM (10 vol) at RT for 18 h to give 1.35 g of 28 as a yellowish oil. Yield:
Quantitative. 1H NMR (300 MHz, CDCl3, δ) 7.14 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz,
2H), 4.65 (s, 2H), 3.59 (m, 4H), 2.85 (m, 4H), 2.82 (set, J = 7.0 Hz, 1H) 1.22 (d, J = 7.0 Hz, 6H).
4-(3,5-Dichlorobenzoyl)-1-(4-i-propylphenoxyacetyl)piperazine (14): prepared from 28 as
described for 21 using 3,5-dichlorobenzoyl chloride (1 eq), TEA (1.2 eq) in DCM (20 mL)
at RT for 3 h. Crystallization of the crude product with 7/3 cyclohexane/ethyl a◦cetate
(5 vol total) at reflux gave 0.95 g of 14 as a white solid. Yield: 42%. M.p: 104.08 C. Tr
(HPLC): 6.67 min. (Method C), Purity = 98.0%. 1H NMR (300 MHz, DMSO-d6, δ) 7.72
(m, J = 1.7 Hz, 1H), 7.50 (m, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 8.1 Hz, 2H), 4.78 (s,
2H), 3.44 (m, 8H), 2.80 (set, J = 6.9 Hz, 1H), 1.14 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6, δ) 166.70, 166.62, 156.48, 141.27, 139.65, 134.76, 129.58, 127.47, 126.09, 114.85,
66.49, 33.02, 24.55 ppm.
4.2. Biological Evaluation
4.2.1. Bacterial Growth Conditions
Staphylococcus aureus strains UAMS-1 is a well-characterized antibiotic-susceptible
osteomyleitus clinical isolate (Blevins Infect. Immun 2003). It was cultured in Mueller
Hinton (MH) broth and processed. as described below. Escherichia coli strain BL21 (DE3)
was cultured in Luria–Bertani (LB) and supplemented with ampicillin (50 µg mL−1).
4.2.2. Antimicrobial Susceptibility Testing
ATCC 29213 and ATCC 43300
The antibacterial activity was tested on both a methicillin-sensitive S. aureus strain
(MSSA, ATCC 29213) and a methicillin-resistant S. aureus strain (MRSA, ATCC 43300). All
of the compounds were dissolved in dimethyl sulphoxide (DMSO). Fresh cell cultures
were used at 105 cells/mL in a final volume of 2 mL. Each bacterial sample was grown
with different compound concentrations at 37 ◦C for 16 h in aerobic culture tubes. The
concentration of bacterial cells was then determined by optical density measurement, at
600 nm (OD600) in a SmartSpecTM 3000 spectrophotometer (Bio-Rad, Oceanside, CA, USA).

Antibiotics 2021, 10, 438
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The presence of antibacterial activity, at any concentration tested, was established for values
of absorbance <0.1 OD600. The MIC was determined as the lowest compound concentration
at which the bacterial cell growth was inhibited.
All of the tests were performed in quadruplicate and, for each series of experiments,
both positive (bacterial cells cultures with no compound) and negative (fresh medium
tubes with compound in the absence of bacteria) controls were included.
4.2.3. RnpA Protein Purification
His-tagged RnpA was purified, as previously described (Eidem AAC 2015). E. coli
BL21 (DE3) cells harboring plasmid pEXP5-nt containing a hexahistidine tag fused to
the N-terminus of S. aureus RnpA coding region were grown in LB supplemented with
50 µ β-d-1-
g mL−1 ampicillin to OD600 = 0.6 and then induced with 1 mM isopropyl
Thiogalactopyranoside (IPTG) for 3 h to induce protein expression. E. coli cells were
◦
subjected to centrifugation at 3.488
×
g for 20 min at 4 C and the cell pellet was resuspended
in 20 mL buffer A (300 mM NaCl, 50 mM Na2HPO4, pH 7.4) containing a mini EDTA-free
protease inhibitor tablet (Roche; Bradford, CT, USA) and 20 mM imidazole. Cells were
mechanically lysed by three passes at 18,000 psi through a French Pressure Cell Press (SLM-
Aminco; Pittsford, NY, USA) and cell debris was removed by centrifugation at 17,000
×
g
for 10 min at 4 ◦C. Supernatants were collected, filtered through a 0.2
µM syringe filter,
and then loaded onto a 5 mL HisPur cobalt column (ThermoFisher Scientific) using the
BioRad Maximizer Duo-Flow Medium Pressure Chromatography System. The protein was
eluted using an imidazole gradient (80 mM to 500 mM). Fractions were assessed for RnpA
presence and purity via SDS-PAGE analysis, Coomassie staining, and Western blotting
using an anti-His antibody (Invitrogen; Grand Island, NY, USA). His-RnpA was eluted
off the column at imidazole concentrations ranging between 200–300 mM. Fractions that
◦
only contained bands corresponding to His-RnpA were pooled and stored at 4 C. No
additional dialysis or concentration protocols were implemented. ptRNA processing assays
were performed with RnpA and ptRNA substrate in the absence of rnpB, as controls to
eliminate the possibility that the protein preparation contained undetected contaminating
RNase proteins. Because ptRNA processing requires the RNase P enzyme (RnpA+rnpB),
no processing activity was detected with RnpA alone, which confirmed the absence of
contaminating RNases (data not shown).
4.2.4. In-Vitro Transcription of RNA
RnpB and RNA substrates (spa, ptRNATyr) for RnpA functional assays, as well as
mature tRNA, were synthesized in vitro, as previously described (Eidem AAC 2015). Each
gene was PCR amplified using S. aureus UAMS-1 chromosomal DNA as a template and the
corresponding oligonucleotide primer pairs, in which the forward primers contained a RNA
polymerase T7 promoter sequence. The following primer pairs were used in this study (T7
promoter underlined): T7-rnpB forward—GAT TAC ATA ATA CGA CTC ACT ATA GGG
TGA TAT TTC GGG TAA TCG CTA TA and rnpB reverse—ACT AGT AGT GAT ATT TCT
ATA AGC CAT G; T7-ptRNATyr forward—GAT TAC ATA A TA CGA CTC ACT ATA GGG
CAC CAT TTA TGG AGG GGT AGC G and ptRNATyr reverse—TGGTGGAG GGGGGCA-
GATTC; T7-spa forward—GAT TAC ATA ATA CGA CTC ACT ATA GGG TTA TAG TTC
GCG ACG ACG TCC AG and spa reverse—TTG AAA AAG AAA AAC ATT TAT TCA
ATT CGT AAA CTA GG. The resulting PCR products were analyzed on a 0.8% agarose gel
and then purified using the Qiagen QIAquick Gel Extraction kit, according to the manufac-
turer’s instructions. In-vitro transcription was performed using the TranscriptAid T7 High
Yield Transcription kit (Fermentas; Burlington, ON, Canada) following the manufacturer’s
instructions. Following in-vitro transcription, the RNA was treated with DNaseI for 90 min
and then re-purified using the Qiagen RNeasy Mini kit. RNA was quantified using a
NanoDrop2000 spectrophotometer.

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4.2.5. In-Vitro ptRNA Processing Assays
S. aureus RNase P activity assays were performed as previously described (Eidem AAC
2015). Briefly, ptRNATyr and rnpB RNA was denatured by heating at 95 ◦C for 5 min and
then slowly cooled to RT. RNA species were then combined with 2
Tris-HCl pH 8.0, 5 mM MgCl2) and incubated for 5 min at 37 C. RNase P was reconstituted
by mixing an equal molar ratio of His-RnpA and rnpB for 15 min at 37 C. Precursor tRNA
×
low salt buffer (50 mM
◦
◦
processing inhibition reactions were performed by combining 5 pmol of reconstituted
RNase P with DMSO (negative control) or compound and incubating for 5 min at 37 ◦C.
5 pmol ptRNATyr was added to each reaction and incubated for an additional 30 min at
◦
37 C. Reactions were stopped with 2
×
RNA loading dye (Thermo Scientific) and heating
at 65 ◦C for 10 min. The samples were electrophoresed on a 7 M urea/8% polyacrylamide
gel and then stained with 0.5 µ
g mL−1 ethidium bromide. The BioRad EZ Gel Doc imaging
system was used to visualize the RNA and the relative abundance of mature tRNATyr in
the DMSO control or in the samples containing the compound. Samples were analyzed
using the BioRad Image Lab densitometry software. The percent inhibitory activity of each
compound was calculated using the following equation: (% experimental processing/%
processing negative control) × 100.
4.2.6. In-Vitro mRNA Degradation Assays
RnpA-mediated degradation was assessed using the RNase Alert QC System (Ther-
mofisher Scientific). His-RnpA (20 pmol of was combined with 0.4
µM (final concentration)
FRET substrate and either DMSO (negative control) or compound (
≤
500 M in 1 RNase
µ
×
Alert Buffer). Reactions were assembled in 96-well PCR plates (BioRad) and incubated
at 37 ◦C for 30 min in the BioRad CFX 96 Connect Real Time instrument, measuring flu-
orescence every 2 min at 490 nm excitation/520 nm emission. Percent inhibition of each
compound was calculated at the 30 min time-point based on the following formula: (RFU
of compound treated RnpA/RFU of DMSO-treated RnpA) × 100.
4.2.7. Cellular tRNATyr Population Measures
Cellular S. aureus tRNATyr pools were measured, as previously described (Colquhoun
Antibiotics 2019). Briefly, S. aureus was grown to OD600 = 0.18 in MHB and then treated with
DMSO (negative control), 0.5
×
or 1
×
MIC of the indicated compound for 1 h with shaking
at 37 ◦C. The culture was removed, combined with an equal volume of acetone:ethanol
(1:1 v/v), and stored at −80 ◦C.
4.2.8. Cellular mRNA Turnover Assays
S. aureus RNA half-life determinations were performed, as described (Eidem AAC
2015). Briefly, S. aureus UAMS-1 was grown to an optical density of 0.18 at 600nm in MHB
and then treated with DMSO (negative control) or 0.5
for 30 min with shaking at 37 ◦C. To inhibit de novo RNA synthesis, rifampin (Alfa Aesar,
Haverhill, MA, USA) was added to the culture at a final concentration of 200
g mL−1
×
MIC of the indicated compound
µ
.
Half of the culture was immediately removed and combined with an equal volume of
ice-cold acetone:ethanol (1:1 v/v). The remainder of the culture was returned to the 37 ◦C
incubator for an additional 5 min and then combined with an equal volume of ice-cold
acetone: ethanol (1:1 v/v) and stored at −80 ◦C.
4.2.9. Bacterial RNA Isolation and Quantitative Reverse Transcription Polymerase Chain
Reaction (qRT-PCR)
Cell suspensions in acetone:ethanol were thawed on ice and centrifuged at 1560× g
for 10 min at 4 ◦C. Acetone:ethanol was decanted and the pellets were air-dried for 5 min
Cell pellets were resuspended in 500
EDTA) and transferred to FastPrep Lysing Matrix B tubes (MP Biomedicals, Santa Ana,
CA, USA). Mixtures were homogenized at 5 m
−1 for 20 s, rested on ice for 5 min, then
homogenized again at 4.5 m
s−1 for 20 s in a FastPrep-24 instrument. Cell lysates were
µL ice-cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM
·
s
·

Antibiotics 2021, 10, 438
20 of 22
◦
g for 15 min at 4 C to remove cell debris. The total bacterial
centrifuged at 16,200
×
RNA and tRNATyr populations were isolated from the supernatant using Qiagen RNeasy
Mini kits and miRNeasy kits, respectively, following the manufacturer’s recommendations
(Germantown, MD, USA). For qRT-PCR, 2
µ
g of RNA substrate was treated with two units
◦
of DNaseI (New England Biolabs, Ipswich, MA, USA) for 1 h at 37 C and then re-purified
using Qiagen RNeasy Mini kits, following the manufacturer’s instructions. RNA was
measured for concentration and quality using a NanoDrop spectrophotometer. Quantabio
(Beverly, MA, USA) qScript cDNA Supermix was used to convert 200 ng of RNA into
cDNA, following the manufacturer’s instructions, which was subsequently amplified using
Quantabio SYBR Green Fast Mix following the manufacturer’s instructions. Fluorescence
was read on the BioRad (Hercules, CA, USA) CFX 96 Connect Real Time Machine. The
transcript levels were compared to the internal control 16S rRNA (∆∆Ct) and plotted
as fold change compared to the control. The following primer pairs were used in this
study for qRT-PCR: spa forward—GCA GAT AAC AAA TTA GCT GAT AAA AAC AT; spa
reverse—CTA ACG CTA ATG ATA ATC CAC CAA ATA C;
−
15 ptRNATyr forward—TTA
ACT GAA TAA GCT GGA GGG G; tRNATyr reverse—TGG TGG AGG GGG GCA GAT TC;
16S rRNA forward—TAA CCT ACC TAT AAG ACT GGG ATA A; 16S rRNA reverse—GCT
TTC ACA TCA GAC TTA AAA A.
4.3. Hotspot Maps
The Fragment Hotspot maps software [28] identifies the location and environment
of binding sites on the protein by first calculating atomic hotspots and then producing
Fragment Hotspot maps while using simple molecular probes. These maps specifically
highlight fragment-binding sites and their corresponding pharmacophores. For this reason
and given the limited knowledge about ligand binding in this target, we applied hotspot
maps to decipher the potential binding site for the set of molecules synthesized in this
work. For this study, we used the crystal structure of S. aureus RNase P protein already
available as 6D1R [26] in the Protein Data Bank.
4.4. Computational Studies
Automated docking was used to assess the potential binding pocket of the set of
molecules together with their binding conformations. A Lamarckian genetic algorithm [29
]
method that was implemented in the program, AutoDock 4.2 [30], was employed. For dock-
ing calculations, Gasteiger charges were added, rotatable bonds were set using AutoDock
tools (ADT), and all of the torsions were allowed to rotate for the ligand. As a first approach,
blind docking was carried out with the reference compounds RNPA2000 and JC1, including
all the receptor as part of the grid to assess the potential binding site of these compounds.
For docking the compounds that were synthesized in this work, we used grid maps with a
grid box size of 60-by-60-by-60 Å3 points and a grid-point spacing of 0.375 Å. The docking
protocol consisted of 100 independent Genetic Algorithm (GA) runs, a population size of
200, and a maximum of 250,000 evaluations, while the other parameters were set as default.
The final best-docked clusters, within the default 2.0-Å root mean square deviation (RMSD),
according to the binding energies and relative population data provided by Autodock,
were analyzed by visual inspection.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/antibiotics10040438/s1, File S1: Supporting Information.
Author Contributions: Conceptualization, L.S. and M.C.; methodology, M.C.; software, V.S.-P.;
validation, L.S., M.C. and C.Z.; formal analysis, E.V.; investigation, L.S. and M.C.; data curation, V.S.
and C.D.G.M.; writing—original draft preparation, L.S.; writing—review and editing, V.S., A.C. and
P.M.D.; supervision, V.S.; project administration, E.V. and P.M.D. All authors have read and agreed to
the published version of the manuscript.
Funding: M.C. is partially supported by the Training Program in Oral Sciences, National Institutes
of Health training grant T90DE021985.

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Institutional Review Board Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Dutescu, I.A.; Hillier, S.A. Encouraging the Development of New Antibiotics: Are Financial Incentives the Right Way Forward? A
Systematic Review and Case Study. Infect. Drug Resist. 2021, 14, 415–434. [CrossRef] [PubMed]
Global Action Plan. Available online: https://www.who.int/antimicrobial-resistance/publications/global-action-plan/en/
(accessed on 12 February 2021).
World Health Organization. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance#:
~{}:text=What%20is%20antimicrobial%20resistance%3F,spread%2C%20severe%20illness%20and%20death (accessed on 12 Febru-
ary 2021).
4.
5.
CDC. Antibiotic Resistance Threats in the United States; Department of Health and Human Services, CDC: Atlanta, GA, USA, 2019.
Aminov, R.I. A brief history of the antibiotic era: Lessons learned and challenges for the future. Front. Microbiol. 2010, 1, 134.
[CrossRef] [PubMed]
6.
7.
8.
Blanchard, C.; Brooks, L.; Beckley, A.; Colquhoun, J.; Dewhurst, S.; Dunman, P.M. Neomycin Sulfate Improves the Antimicrobial
Activity of Mupirocin-Based Antibacterial Ointments. Antimicrob. Agents Chemother. 2016, 60, 862–872. [CrossRef] [PubMed]
Rungelrath, V.; DeLeo, F.R. Staphylococcus aureus, Antibiotic Resistance, and the Interaction with Human Neutrophils. Antioxid.
Redox Signal. 2021, 34, 452–470. [CrossRef]
Straniero, V.; Suigo, L.; Casiraghi, A.; Sebastián-Pérez, V.; Hrast, M.; Zanotto, C.; Zdovc, I.; De Giuli Morghen, C.; Radaelli, A.;
Valoti, E. Benzamide Derivatives Targeting the Cell Division Protein FtsZ: Modifications of the Linker and the Benzodioxane
Scaffold and Their Effects on Antimicrobial Activity. Antibiotics 2020, 9, 160. [CrossRef]
9.
Holmes, N.E.; Johnson, P.D.R.; Howden, B.P. Relationship between vancomycin-resistant Staphylococcus aureus, vancomycin-
intermediate S. aureus, high vancomycin MIC, and outcome in serious S. aureus infections. J. Clin. Microbiol. 2012, 50, 2548–2552.
[CrossRef] [PubMed]
10. Foster, T.J. Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbiol. Rev. 2017, 41,
430–449. [CrossRef] [PubMed]
11. Li, Y.; Yang, L.; Fu, J.; Yan, M.; Chen, D.; Zhang, L. Microbial pathogenicity and virulence mediated by integrons on Gram-positive
microorganisms. Microb. Pathog. 2017, 111, 481–486. [CrossRef]
12. Solomon, S.L.; Oliver, K.B. Antibiotic Resistance Threats in the United States: Stepping Back from the Brink. AFP 2014, 89,
938–941.
13. McGuinness, W.A.; Malachowa, N.; DeLeo, F.R. Vancomycin Resistance in Staphylococcus aureus. Yale J. Biol. Med. 2017, 90,
269–281.
14. Straniero, V.; Sebastián Pérez, V.; Hrast, M.; Zanotto, C.; Casiraghi, A.; Suigo, L.; Zdovc, I.; Radaelli, A.; De Giuli Morghen, C.;
Valoti, E. Benzodioxane-benzamides as antibacterial agents: Computational and SAR studies to evaluate the influence of the
7-substitution in FtsZ interaction. ChemMedChem 2020, 2, 195–209. [CrossRef] [PubMed]
15. Wenzel, R.P. The antibiotic pipeline—Challenges, costs, and values. N. Engl. J. Med. 2004, 351, 523–526. [CrossRef] [PubMed]
16. Mitchell, P.; Petfalski, E.; Shevchenko, A.; Mann, M.; Tollervey, D. The Exosome: A Conserved Eukaryotic RNA Processing
Complex Containing Multiple 3’→5’ Exoribonucleases. Cell 1997, 91, 457–466. [CrossRef]
17. Tejada-Arranz, A.; de Crécy-Lagard, V.; de Reuse, H. Bacterial RNA Degradosomes: Molecular Machines under Tight Control.
Trends Biochem. Sci. 2020, 45, 42–57. [CrossRef] [PubMed]
18. Wang, X.; Wang, C.; Wu, M.; Tian, T.; Cheng, T.; Zhang, X.; Zang, J. Enolase binds to RnpA in competition with PNPase in
Staphylococcus aureus. FEBS Lett. 2017, 591, 3523–3535. [CrossRef]
19. Roux, C.M.; DeMuth, J.P.; Dunman, P.M. Characterization of components of the Staphylococcus aureus mRNA degradosome
holoenzyme-like complex. J. Bacteriol. 2011, 193, 5520–5526. [CrossRef]
20. Olson, P.D.; Kuechenmeister, L.J.; Anderson, K.L.; Daily, S.; Beenken, K.E.; Roux, C.M.; Reniere, M.L.; Lewis, T.L.; Weiss, W.J.;
Pulse, M.; et al. Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial
Activity, and Attenuate Pathogenesis. PLoS Pathog. 2011, 7, e1001287. [CrossRef]
21. Carpousis, A.J. The RNA degradosome of Escherichia coli: An mRNA-degrading machine assembled on RNase E. Annu. Rev.
Microbiol. 2007, 61, 71–87. [CrossRef]
22. Eidem, T.M.; Lounsbury, N.; Emery, J.F.; Bulger, J.; Smith, A.; Abou-Gharbia, M.; Childers, W.; Dunman, P.M. Small-molecule
inhibitors of Staphylococcus aureus RnpA-mediated RNA turnover and tRNA processing. Antimicrob. Agents Chemother. 2015, 59,
2016–2028. [CrossRef]
23. Lounsbury, N.; Eidem, T.; Colquhoun, J.; Mateo, G.; Abou-Gharbia, M.; Dunman, P.M.; Childers, W.E. Novel inhibitors of
Staphylococcus aureus RnpA that synergize with mupirocin. Bioorg. Med. Chem. Lett. 2018, 28, 1127–1131. [CrossRef]
24. Colquhoun, J.M.; Ha, L.; Beckley, A.; Meyers, B.; Flaherty, D.P.; Dunman, P.M. Identification of Small Molecule Inhibitors of
Staphylococcus aureus RnpA. Antibiotics 2019, 8, 48. [CrossRef] [PubMed]
25. Naclerio, G.A.; Abutaleb, N.S.; Li, D.; Seleem, M.N.; Sintim, H.O. Ultrapotent Inhibitor of Clostridioides difficile Growth, Which
Suppresses Recurrence In Vivo. J. Med. Chem. 2020, 63, 11934–11944. [CrossRef] [PubMed]

Antibiotics 2021, 10, 438
22 of 22
26. Ha, L.; Colquhoun, J.; Noinaj, N.; Das, C.; Dunman, P.M.; Flaherty, D.P. Crystal structure of the ribonuclease-P-protein subunit
from Staphylococcus aureus. Acta Crystallogr. F Struct. Biol. Commun. 2018, 74, 632–637. [CrossRef] [PubMed]
27. DeBons, F.E.; Loudon, G.M. Protected diaminomethane. J. Org. Chem. 1980, 45, 1703–1704. [CrossRef]
28. Radoux, C.J.; Olsson, T.S.G.; Pitt, W.R.; Groom, C.R.; Blundell, T.L. Identifying Interactions that Determine Fragment Binding at
Protein Hotspots. J. Med. Chem. 2016, 59, 4314–4325. [CrossRef] [PubMed]
29. Morris, G.M.; Goodsell, D.S.; Halliday, R.S.; Huey, R.; Hart, W.E.; Belew, R.K.; Olson, A.J. Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 1998, 19, 1639–1662. [CrossRef]
30. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4:
Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785–2791. [CrossRef]