Mechanistic Studies and In Vivo Efficacy of an Oxadiazole-Containing Antibiotic

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections are still difficult to treat, despite the availability of many FDA-approved antibiotics. Thus, new compound scaffolds are still needed to treat MRSA. The oxadiazole-containing compound, HSGN-94, has been shown to reduce lipoteichoic acid (LTA) in S. aureus, but the mechanism that accounts for LTA biosynthesis inhibition remains uncharacterized. Here, we report the elucidation of the mechanism by which HSGN-94 inhibits LTA biosynthesis via utilization of global proteomics, activity-based protein profiling, and lipid analysis via multiple reaction monitoring (MRM). Our data suggest that HSGN-94 inhibits LTA biosynthesis via direct binding to PgcA and downregulation of PgsA. We further show that HSGN-94 reduces the MRSA load in skin infection (mouse) and decreases pro-inflammatory cytokines in MRSA infected wounds. Collectively, HSGN-94 merits further consideration as a potential drug for staphylococcal infections.

Introduction:

Staphylococcus aureus is one of the major causative agents of community- and hospital-acquired bacteremia, surgical site infections, osteomyelitis, pneumonia, and skin infections¹. Methicillin-resistant S. aureus (MRSA) bacteremia causes higher death rates than infections caused by non-MRSA strains^2,3. Although there are several antibiotics used to treat MRSA such as clindamycin, minocycline, daptomycin or vancomycin⁴, drug-resistance has been documented, attributing to the fact that 14% of patients who get serious MRSA infections die^4–8. Additionally, newer antibacterial agents developed to treat drug-resistant bacterial pathogens like MRSA are only derivatives of existing drugs. Thus it is likely that resistance mechanisms affecting the older drugs would alter the activity of the newer ones⁹. The need for novel chemical entities with new mechanisms of action cannot be overstated.

The membrane of many Gram-positive bacteria, such as Streptococcus pneumonia and S. aureus, is decorated with an anionic 1,3-glycerolphosphate-containing polymer called LTA (lipoteichoic acid)¹¹. In S. aureus, LTA is important for bacterial growth¹⁰, cell wall physiology¹², membrane homeostasis¹³, virulence^14–15, and biofilm formation¹⁶. LTA synthesis begins in the cytoplasm where the α-phosphoglucomutase (PgcA) synthesizes glucose-1-phosphate from glucose-6-phosphate. Next, uridyltransferase GtaB produces uridyl diphosphate-glucose (UDP-glucose) via the activaton of uridyl triphosphate (UTP). Then, YpfP transports glucose from UDP-Glucose to diacylglycerol (DAG) to give diglucosyl-diacylglycerol (Glc₂-DAG). Glc₂-DAG is flipped to membrane by the flippase LtaA where it then acts as a starting unit for LTA. Lastly, the synthase LtaS catalyzes the addition of glycerol phosphate to Glc₂-DAG, thereby generating LTA^{10, 17} (Figure 1A). The mechanism by which LtaS generates LTA involves the use of catalytic threonine (T300)^18–20. Phosphatidyl glycerol (PG) transfers phosphoglycerol units to T300, realeasing DAG to produce a covalent intermediate. Phosphoglycerol is then transferred to Glc₂-DAG via reaction with the covalent intermediate to give glycerol-phosphate- diglucosyl-diacylglycerol (GroP-Glc₂DAG). Repeat units are then added to the glycerol tip, giving rise to LTA (Figure 1B). Since LTA is essential to S. aureus, it has been deemed a potential antimicrobial target²¹.

Figure 1.

LTA biosynthesis in S. aureus involves both Glc₂DAG and PG. (A) PgcA synthesizes glucose-1-phosphate from glucose-6-phosphate. Next, UDP-glc is produced from GtaB’s activation of UTP. YpfP then transfers glucose from UDP-Glc to DAG in order to make Glc₂-DAG. Then, LtaA flips Glc₂-DAG to the outer membrane where LtaS catalyzes the addition of glycerol phosphate to Glc₂-DAG, generating LTA¹⁰. (B) LtaS synthesizes LTA via utilization of catalytic T300. PG transfers phosphoglycerol units to T300 formig a covalent intermediate. Then, phosphoglycerol units are transferred to Glc₂DAG through a reaction with the covalent intermediate, giving rise to GroP-Glc₂DAG. Repeat units are then added to the polymer, producing LTA. (C) HSGN-94 is a highly potent antimicrobial agent discovered to inhibit LTA biosynthesis.

Our lab has demonstrated that N-(1,3,4-oxadiazol-2-yl)benzamides are effective antimicrobial agents^22–27. We previously demonstrated that the N-(1,3,4-oxadiazol-2-yl)benzamide HSGN-94 was an antimicrobial agent against drug-resistant Gram-positive bacteria²⁷. The MIC (minimum inhibitory concentrations) of HSGN-94 against bacteria ranged from 0.25 μg/mL to 2 μg/mL²⁷ (Figure 1C). Additionally, HSGN-94 was found to be an inhibitor of LTA biosynthesis in S. aureus²⁷. We also demonstrated HSGN-94’s ability to inhibit MRSA and vancomycin-resistant Enterococci biofilm formation²⁶.

Building upon our previous studies, this study investigated the antibacterial profile of HSGN-94 including structure-activity relationship studies, MICs against various multidrug-resistant Gram-positive bacteria, cytotoxicity assessment, and the resistance development assay. We also elucidated the mechanism by which HSGN-94 inhibits LTA biosynthesis in S. aureus, utilizing several mechanistic studies, including global proteomics, activity-based protein, and transcriptional profiling, lipidomics, and macromolecular synthesis inhibition. Finally, we evaluated HSGN-94’s in vivo efficacy in a MRSA murine wound infection model as well as its ability to inhibit the manifestation of pro-inflammatory cytokines and the effect of HSGN-94’s treatment on the histopathological features of the mice skin.

Results and Discussion:

Structure-activity relationship studies

We established structure-activity-relationship (SAR) studies of HSGN-94, which has indicated important structural features of the molecule that account for its potent antibacterial activity. The synthetic schemes of these new analogs can be found in the supporting information (SI; Schemes S1-S8). The results of the initial screening of the synthesized analogs against S. aureus are included in Table S1. We discovered that the size of the sulfonamide ring A (Figure 2, Series 1) and nature of substitution were all critical for antibacterial activity. For instance, while HSGN-94 (3,5-dimethylpiperidine) had an MIC of 0.25 μg/mL (0.5 μM), the analog 2-azabicyclo [2.2.1]heptane (5) had an MIC value of 2 μg/mL (4.1 μM). Furthermore, monomethyl substituted analogs 6, 7, and 8, also had less antibacterial activity with MICs of 1 μg/mL (2 μM), 2 μg/mL (4 μM), and 2 μg/mL (4 μM) respectively. Analogs containing smaller ring systems (1, 2, 3, and 4) showed less activity than HSGN-94 as well, with MIC values from 2 μg/mL (4.2 μM) to 16 μg/mL (36.3 μM) (Table S1).

Figure 2.

Summary of structure-activity-relationship study of sulfonamide-containing N-(1,3,4-oxadiazol-2-yl)benzamides

Moreover, we determined that an unsubstituted phenyl moiety of the benzamide (ring B) was optimal for the antibacterial activity, as replacing ring B with other heterocycles (Figure 2, Series 2) or substituting ring B with electron-donating or electron-withdrawing groups (Figure 2, Series 3) showed a decrease in the antibacterial activity by 4 to >256 times (Table S1). Likewise, both the amide bond and trifluoromethylphenyl group (ring C) were imperative for antibacterial activity. Conversion of the amide to a tertiary amide (17) or reversion of the amide (18) completely abrogated the anti-S. aureus activity (MICs of >64 μg/mL). Substitution of ring C with alkyl-containing groups (19 and 20) showed a reduction in antibacterial activity (MICs = 64 - >64 μg/mL) (Table S1). Altogether, our SAR investigations demonstrated that HSGN-94 displayed the most potent activity against staphylococci. Therefore, it was selected for further characterization.

Profiling the antibacterial activity of HSGN-94

We explored the antibacterial activity of HSGN-94 versus a panel of multidrug-resistant bacterial strains, such as methicillin-sensitive, methicillin-, and vancomycin-resistant S. aureus, S. epidermidis, Streptococcus pneumonia, S. pyogenes, vancomycin-resistant Enterococcus faecium and E. faecalis, and Listeria monocytogenes. As reported in our study before²⁷, HSGN-94 showed potent activity against staphylococcal strains with MIC values ranging from 0.25 μg/mL (0.5 μM) to 1 μg/mL (2 μM) (Table S2). HSGN-94 was also found to be highly potent to drug-resistant S. pneumoniae and S. pyogenes as well (MICs ranging from 0.06 μg/mL (0.1 μM) to 0.25 μg/mL (0.5 μM), surpassing the activity of linezolid and vancomycin (Table S3). S. pneumoniae is a pathogen that produces a variety of infections such as ear and sinus infections as well as pneumonia, bloodstream infections, and meningitis^28–29. Additionally, S. pneumoniae causes around 2 million infection in the United States annually, resulting in more than 6,000 deaths as well as $4 billion in health-care costs. Additionally, greater than 30% of pneumococcal infections are resistant to FDA approved antibiotics^30–31. Thus, the CDC classifies S. pneumoniae as a serious threat and new agents against this pathogen are needed³¹. Additionally, the compound maintained the same potency against vancomycin-resistance enterococci (MIC = 0.25 μg/mL (0.5 μM)), a serious threat pathogen that greatly demands new therapeutics^32–34. HSGN-94 was also superior to linezolid and vancomycin against L. monocytogenes with an MIC of 0.06 μg/mL (0.1 μM) (Table S3).

The cytotoxicity profile of HSGN-94 was assessed against human keratinocyte (HaCaT) cells (Figure S1). The compound was non-toxic to HaCaT cells at a concentration up to 64 μg/mL, which represents 64 to 256 times its MIC values against staphylococcal strains.

Moreover, we assessed MRSA USA300’s ability to form resistance to HSGN-94, using the multi-step resistance selection assay. Excitingly, the compound displayed low propensity to develop resistance to MRSA USA300, where its MIC remained unchanged over 65 passages (Figure S2). In contrast, MRSA formed rapid resistance to ciprofloxacin where its MIC progressively increased after following passages reaching 128-fold increase in the MIC after 65 passages (Figure S2).

Effects of HSGN-94 on global proteomics in S. aureus:

After recognizing that obtainment of HSGN-94 resistant mutants via serial passaging was not possible (Figure S2), we went ahead and utilized global proteomics to ascertain the proteins that are impacted after S. aureus treatment with HSGN-94. S. aureus was treated with HSGN-94 for 2 hours and then the total protein content was isolated for investigation via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples treated with HSGN-94 were related to samples treated only with DMSO (control). Proteomics data was sorted using label-free quantitation (LFQ) and analysis was pursued for proteins showing LFQ response in all 3 samples tested. During the evaluation of the proteomics data, we discovered a total number of 1475 proteins. Yet, out of these proteins, 1431 proteins (97.0%) were found to be common to both DMSO- and HSGN-94-treated samples, while 30 proteins (3.0%) were only detected in DMSO-treated sample, and 14 proteins (1.0%) were only found in HSGN-94-treated sample (Figure 3A). Stringent filtration using statistical analysis of the 1475 total proteins (where p <0.05) gave 423 proteins, which demonstrated that the treatment and control samples gathered into two differentially expressed groups (Figure 3B). From those 423 significant proteins, 198 were downregulated, while 225 were upregulated. We then filtered this data set using p-value (p) and fold change (Log₂FC). Based on this analysis, 18 significant proteins were downregulated by Log₂FC ≤ −2, whereas 7 proteins were upregulated by Log₂FC ≥ 2 after HSGN-94 treatment (Figure 3B & 3C).

Figure 3.

Global proteomics assessment of S. aureus cells after HSGN-94 treatment. (A) A Venn diagram was constrcuted to compare proteins found in DMSO-treated cells individually, HSGN-94-treated cells individually and in both groups. (B) Heatmap analysis of significant proteins (p <0.05) demonstrating differential expression between both DMSO- and HSGN-94-treated S. aureus. (C) Volcano plot of significant proteins displaying the Log₂ fold change (Log₂FC, x-axis) vs. p-value (y-axis). Most down and upregulated proteins have been labled. Differentially expressed proteins were identified as p ≤0 .05 and Log₂FC ≤ −2 for downregulated proteins or p ≤ 0.05 and Log₂FC ≥ 2 for upregulated ones. The Perseus software was used to analyze data³⁵. OriginPro 2017 Software (OriginLab, Massachusetts, USA) was utilized to construct the volcano plot.

Evaluation of the 18 downregulated proteins (those with Log₂FC ≤ −2) demonstrated that S. aureus treatment with HSGN-94 impacted several bacterial processes such as transcription, translation, nucleotide metabolism, amino acid biosynthesis, and carbohydrate biosynthesis (Table 1). Importantly, HSGN-94 seemed to have the most significant impact on virulence (Figure S3 and Table 1). For instance, thermonuclease (Nuc) which is an important virulence factor in S. aureus as it is vital for DNA and RNA degradation, a crucial part of the organism’s denfense mechanism^36–37, was the most downregulated protein (Log₂FC = −5.1) (Figure S3 and Table 1). Likewise, other virulence factors were also downregulated such as gamma-hemolysin subunit B, LukS-PV (Log₂FC = −3.7), leucotoxin LukD (Log₂FC = −2), Nitrate reductase subunit alpha NarG (Log₂FC = −2), Lipoyl synthase LipA (Log₂FC = −2), and hydrolase SdrD (Log₂FC = −2) (Figure S3 and Table 1). Additionally, HSGN-94 appears to substantially affect the type VII secretion system (T7SS) of S. aureus, which is an essential pathway for bacterial virulence³⁸. The T7SS, activated by host’s lipids, consists of four membrane-associated proteins (EsaA, EssA, EssB, and EssC), three cytosolic proteins (EsaB, EsaE, and EsaG), and five virulence factors that are produced (EsxA, EsxC, EsxB, EsxD, and EsaD)^39,40. S. aureus treatment with HSGN-94 showed downregulation of several proteins in the T7SS pathway like EsaA (Log₂FC = −2.0), EsxA (Log₂FC = −2.0), and EssB (Log₂FC = −3.3) (Figure S3 and Table 1). Similarly, both EssC and EsaB were also downregulated by HSGN-94 treatment as they were only identified in the DMSO-treated group (Table 2).

Table 1.

Proteins that were downregulated in S. aureus after HSGN-94 treatment.

ID	Protein	Classification	Log2FC	p-value
Virulence
gi|685631952	Nuc	Thermonuclease	−5.1	0.0010
gi|685633550	LukS-PV	Gamma-hemolysin subunit B	−3.7	0.000026
gi|685631441	EssB	Type VII secretion protein EssB	−3.3	0.024
gi|685631437	EsxA	Type VII secretion protein EsxA	−2.0	0.00063
gi|685631438	EsaA	Type VII secretion protein EsaA	−2.0	0.00011
gi|685631717	SdrD	Hydrolase	−2.0	0.00010
gi|685632604	LukD	Leucotoxin LukD	−2.0	0.013
gi|685633523	NarG	Nitrate reductase subunit alpha	−2.0	0.0021
gi|685631988	LipA	Lipoyl synthase	−2.0	0.000029
Nucleotide metabolism
gi|685632728	GloB	Hydroxyacylglutathione hydrolase	−2.0	0.000081
gi|685632295	PyrF	Orotidine 5-phosphate decarboxylase	−2.2	0.00010
gi|685632294	CarB	Carbamoyl-phosphate synthase large chain	−2.0	0.000046
Transcription
gi|685633415	SarR	MarR family transcriptional regulator	−2.0	0.000014
Translation
gi|685632759	MtaB	30S ribosomal protein S12 methylthiotransferase	−2.0	0.0067
Amino acid biosynthesis
gi|685633333	Als	Acetolactate synthase	−2.7	0.0031
gi|685633124	DapE	Succinyl-diaminopimelate desuccinylase	−3.3	0.000044
gi|685632550	IlvA	Threonine dehydratase	−2.0	0.0030
Carbohydrate metabolism
gi|685631305	AdhE	Acetaldehyde dehydrogenase	−2.5	0.0039

Table 2.

Proteins that were upregulated in S. aureus after treatment with HSGN-94.

ID	Protein	Classification	Log2FC	p-value
Metabolism
gi|685633108	Bsh	choloylglycine hydrolase	3.6	0.0011
gi|685631431		peptidase M23	2.0	0.000037
gi|685631436	TraG	CHAP domain-containing protein	2.0	0.000010
gi|685632306	DefA	peptide deformylase	2.0	0.0075
Iron Regulation
gi|685631322	IsdI	Heme oxygenase (staphylobilin-producing) 2	3.20	0.00014
Glycolysis
gi|685633543	GpmA	2,3-bisphosphoglycerate-dependent phosphoglycerate mutase	2.2	0.00000055
Proteolysis
gi|685632066	MecA	adaptor protein MecA	2.0	0.000077

Furthermore, we also analyzed the 30 proteins that were only detected in the DMSO-treated group (see figure Figure 3A and Table 3). Detection of these proteins in the DMSO group only indicates that they were massively downregulated by HSGN-94. In this group of proteins, we utilized the report by Charles et al⁴⁰ that provided a thorough listing of S. aureus essential genes to identify essential proteins that were only found in the DMSO-treated group. We discovered that HSGN-94 downregulated CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase (PgsA). PgsA is an essential protein in S. aureus because of its important role in phospholipid synthesis in the bacteria⁴¹. Additionally, PgsA shares 29% sequence identity with the human cardiolipid synthase which is responsible for the conversion of cytosine diphosphate diacylglycerol (CDP-DAG) and PG to cardiolipin and cytosine monophosphate (CMP) in mitochondria⁴².

Table 3.

Proteins found only in DMSO-treatment group and considered downregulated by HSGN-94 treatment

Protein	Biological process
DNA recombination protein RecF	Homologous recombination
PTS mannose transporter subunit IIABC	Mannose uptake
glycerophosphodiester phosphodiesterase	lipid metabolism
copper-translocating P-type ATPase	copper transport
oxidoreductase
DNA-binding response regulator	Transcription
molybdenum ABC transporter ATP-binding protein	Molybdate transport
threonine synthase	threonine biosynthesis
nucleoside permease	nucleoside intramembrane transporter
sodium ABC transporter permease
CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase	Phosphatidylglycerol (PG) synthesis
aureolysin	Virulence factor
carboxylesterase	Metabolism
type VII secretion protein EsaB	Virulence factor
hydrolase	Metabolism
signal peptidase II	lipoprotein biosynthesis
peptidase	Metabolism
sodium ABC transporter ATP-binding protein	sodium transport
MAP domain-containing protein	Virulence factor
protein EssC	Virulence factor
nitrite reductase (NAD(P)H) small subunit	Nitrate assimilation
glutamyl endopeptidase	Virulence factor
50S rRNA methyltransferase	Translation
multifunctional 2,3-cyclic-nucleotide 2-phosphodiesterase/5-nucleotidase/3-nucleotidase	Nucleotide catabolism
site-specific integrase
holo-ACP synthase	Pantothenate and CoA biosynthesis
Membrane protein
uroporphyrinogen-III C-methyltransferase	Porphyrin and chlorophyll metabolism
peptide ABC transporter permease
orthopoxovirus protein, PF05708 family	hydolase

In S. aureus, PgsA synthesizes PG-P from CDP-DAG by exhanging glycerol-3-phosphate (Gro-3-P) for CMP. Then, phosphatidylglycerol phosphate phosphatase (PgpP) rapdily dephosphorylates PG-P to give PG. Next, cardiolipin synthase (Cls) synthesizes cardiolipin (CL) via the condensation of two PG molecules⁴³. Additionally, as mentioned above, LtaS also uses PG to synthesize LTA. PgsA is essential for the synthesis of PG and PG is important for LTA biosynthesis (Figure 4A). Next we sought to confirm that HSGN-94 downregulates PgsA via utilization of RT-qPCR. These results showed that after HSGN-94 treatment, the tested S. aureus cells exhibited a reduction in pgsA gene expression (Figure 4B), which confirmed our global proteomics analysis.

Figure 4.

HSGN-94 downregulates PgsA. (A) PgsA synthesizes phosphatidylglycerol phosphate (PG-P) from cytosine diphosphate diacylglycerol (CDP-DAG) by exhanging glycerol-3-phosphate (Gro-3-P) for cytosine monophosphate (CMP). Phosphatidylglycerol phosphate phosphatase (PgpP) rapdily dephosphorylates PG-P to give PG. Cardiolipin synthase (Cls) synthesizes cardiolipin (CL) via the condensation of two PG molecules. LtaS also uses PG in order to synthesize LTA. (B) The effect of S. aureus treated with HSGN-94 (0.25 μg/mL) pgsA expression. Experiments were performed in triplicate and normalized with 16S RNA. Error bars represent standard-deviation. Statistical significance amongst both DMSO- and HSGN-94-treated groups were established by Student’s t-test assessment (unpaired, two-tailed) and is characterized as *p ≤ 0.05.

While evaluating proteins that upregulated by HSGN-94 treatment, we discovered that the compound played a significant role in iron acquisition in S. aureus. For example, S. aureus treated with HSGN-94 resulted in upregulation of IsdI, IsdE, HarA, SbnA, SbnC, SbnF, and SbnE, proteins associated with iron obtainment (Table 2 and Table 4). HarA, IsdI, and IsdE are all part of the Isd system in S. aureus found within the cell wall, which facilitates iron acquisition⁴⁴. Specifically, HarA binds heme within the cell wall and travels along the near iron transporter (NEAT) domains of IsdC and IsdA to IsdE resulting in the introduction of heme in the cytoplasm. Then, heme is degraded by IsdI which releases available iron to fulfill nutrient needs⁴⁴. HrtA is a protein that regulates heme transportation into the cell and is a component of the heme-regulated transport (Hrt) system⁴⁵. SbnA, SbnC, SbnF, and SbnE are all part of the sbn operon which are important for the synthesis of the siderophore staphyloferrin B⁴⁴.

Table 4.

Proteins found only in HSGN-94-treatment group and considered upregulated

Protein	Biological process
zinc ABC transporter ATP-binding protein	Zinc transport
siderophore biosynthesis protein SbnE	iron acquistion
DUF4930 domain-containing protein	metabolism
glycine cleavage system protein H	glycine metabolism
thiol reductase thioredoxin	cell redox/ glycerol ether metabolic process
Cro/Cl family transcriptional regulator	Transcription
23S rRNA (pseudouridine(1915)-N(3))-methyltransferase RlmH	Translation
L-serine dehydratase, iron-sulfur-dependent subunit alpha	Amino acid metabolism/gluconeogenesis
CHAP domain-containing protein	Pentaglycine cleavage
acylphosphatase	Pyruvate metabolism
heme uptake system protein IsdE	iron acquisition
siderophore biosynthesis protein SbnC	iron acquisition
dihydroxyacetone kinase subunit L	glycerol metabolism (Glycerone phoshorlyation)
haptoglobin-binding heme uptake protein HarA	iron acquisition
ribonuclease HIII	DNA replication, cleave RNA-DNA hybrids
siderophore biosynthesis protein SbnA	iron acquisition
siderophore biosynthesis protein SbnF	iron acquisition
30S ribosomal protein S20	translation

HSGN-94 Inhibits Glc₂-DAG in S. aureus:

The global proteomics analysis demonstrated that HSGN-94 had an effect on numerous biological processes in S. aureus (Figure S3 and Table 1) and the levels of several proteins (~80) were impacted by HSGN-94. However global proteomics does not reveal the actual binding protein(s) to the compound so we further performed activity-based protein profiling to identify the putative targets of HSGN-94^46–49. An affinity probe (HSGN-probe) was synthesized in 3 steps via click chemistry between the biotin azide and an alkynyl oxadiazole benzamide (I11) (Figure 5B). Then, a pull-down assay was performed. To ensure that proteins being captured with the HSGN-Probe also bind the unlabeled compound HSGN-94, we used a competition strategy, vide infra, to identify proteins that putatively bind to HSGN-94. Briefly, S. aureus ATCC 25923 cells (at the exponential phase) were incubated with HSGN-Probe plus 50 μM HSGN-94 (sample A) or with HSGN-Probe only (sample B) for 4 hours at 25°C with gentle agitation (Figure 5B). Then, cells were lysed and the biotinylated probes were captured with steptavidin beads, and enriched proteins were evalauted via SDS-PAGE. The bands that show on sample A lane but not on sample B lane (i.e. unique to the HSGN-probe without HSGN-94 competition) would be the proteins that bind to HSGN-94.

Figure 5.

Probe Synthesis and Pull-Down Assay. (A) Synthesis of HSGN-Probe^a. ^aReagents and Conditions: (a) MeOH, rt, 12 h 53% (b) BOP Reagent, DIPEA, DMF, rt, 12 h, 24% (c) CuSO₄·5H₂O, Na Ascorbate, DMF:H₂O (10:1), 60°C, 12 h, 77%. (B) Schematic of pull-down assay experiment to identify target(s) of HSGN-94.

Phosphoglucomutase (PgcA) was one of the proteins unique to HSGN-Probe group (Table S4). PgcA is found in both bacteria as well as mammalian cells. However, it has been shown that the structure of bacterial PgcA shares just 25% amino acid identity with mammalian PgcA^50–51. In S. aureus, PgcA synthesizes glucose-1-phosphate from glucose-6-phosphate (the first step in the LTA biosynthesis pathway, Figure 1A) which later gives rise to Glc₂-DAG. Thus, since HSGN-94 inhibits PgcA, it would also inhibit Glc₂-DAG formation. To test this hypothesis, we treated S. aureus ATCC 25923 with HSGN-94 (0.25×, 1×, and 8×MIC) for 5 hours and then extracted total membrane lipids using a similar procedure outlined by Scheewind et al⁵². The lipids were then separated by thin layer chromatography (TLC), and imaged by α-naphthol/sulfuric acid staining. The TLC indicated that HSGN-94 treatment reduced the amount of the α-naphthol-reactive species, which we tentatively assigned as Glc₂-DAG and further characterized (Figure 6A). To identify the stained species, lipids were isolated from TLC plates and examined by MALDI-TOF mass spectrometry. The mass-to-charge (m/z) ratio of the major ion signal of the stained species agreed with the expected mass of Glc₂-DAG [M + H]⁺ containing C₁₅ to C₁₈ fatty acids chain lengths (Figure 6B). For instance, the expected m/z for the [M + H]⁺ of Glc₂-DAG bearing C₁₈ and C₁₅ fatty acid chains was 907.63 and the observed m/z for DMSO and HSGN-94 treated samples were the same as that of the projected m/z (907.62–907.63) (Figure 6B and SI for mass spectra). In summary, our results reveal that, compared to DMSO-treated S. aureus, HSGN-94 (0.25×, 1×, and 8×MIC) inhibits Glc₂-DAG which we hypothesize is a result of HSGN-94’s direct binding to PgcA.

Figure 6.

HSGN-94 inhibits Glc₂-DAG. (A) Evaluation of S. aureus glycolipids via TLC. Membrane lipids were isolated from S. aureus ATCC 25923, treated with either DMSO (control) or HSGN-94 (0.25×, 1×, and 8×MIC). (B) Glycolipids were separated from TLC and assessed via MALDI mass spectoscopy.

Multiple reaction monitoring profiling (MRM-Profiling) of lipids demonstrates HSGN-94 selectively affects phosphaditlyglycerol (PG) in S. aureus

It has been demonstrated that even in the absence of Glc₂-DAG, LTA can still be formed as LtaS uses PG as an alternative starter unit¹². Polymers formed on PG to make the alternative PG-LTA are much longer than polymers formed on Glc₂-DAG^{12, 52}. Additionally, it was reported that S. aureus cells that make these longer polymers have cell division defects^{12, 53}, are less virulent^{15, 52}, and are more sensitive to β-lactam antibiotics as well as other cell envelope stresses¹². However, since the inhibition of Glc₂-DAG would not completely deplete LTA, and our results indicte that HSGN-94 potently depletes LTA from S. aureus,²⁷ we hypothesized there must be another mechanism by which HSGN-94 inhibits LTA biosynthesis. Since global proteomics and RT-qPCR show that HSGN-94 downregulates pgsA, an essential protein in S. aureus for PG synthesis, we wondered if HSGN-94 also had an effect on PG. To determine this, we proceeded to perform multiple reaction monitoring profiling (MRM-Profiling)⁵⁴ to differentiate lipid profile distinctions among S. aureus ATCC 25923 treated with either DMSO (control), or HSGN-94 (1×, and 8× MIC) for 5 hours. Lipids were extracted and analyzed using using electrospray ionization mass spectroscopy (ESI-MS). MRMs were refined by ion counts and false discovery rate (FDR) modified p-value in ANOVA, and then investigated using principal component analysis (PCA). Our experiment focused on analyzing differences in glycerophospholipids and total membrane lipids in S. aureus treated cells. After obtaining this data, PCA scores plots were generated to evaluate HSGN-94’s effects on PG (Figure 7A), total membrane lipids (Figure 7B), phosphotitdylcholine (PC) (Figure 7C), and phosphatidylethanolamine (PE) (Figure 7D). Each point in the PCA plot signifies a single sample of lipid extract from the staphyloccocci and the shaded oval region is characterized as the calculated 95% confidence region for every group. Interestingly, we discovered that HSGN-94 seemed to affect PG (Figure 7A). However, HSGN-94 did not affect total lipids, PC or PE (Figures 7B–D).

Figure 7.

PCA scores plots of S. aureus treated with either DMSO (control) or HSGN-94 ((1×, and 8× MIC). (A) Effects on PG synthesis. (B) Effects on total membrane lipids. (C) Effects on PC synthesis. (D) Effects on PE synthesis. *Note: Blue = DMSO, Red = 1× MIC HSGN-94, and Green = 8× MIC HSGN-94.

Proposed mechanism for HSGN-94’s inhibition of LTA biosynthesis

Since our data demonstrates that HSGN-94 directly binds to PgcA as well as downregulates pgsA, we hypothesized that HSGN-94 inhibits LTA biosynthesis in two distinct manners (Figure 8). First, HSGN-94 directly binds to PgcA thereby inhibiting the synthesis of Glc₂-DAG. Secondly, HSGN-94 downregulates PgsA expression, which causes an effect on PG synthesis. We currently do not know if the inhibition of Glc₂-DAG synthesis has an indirect effect on PgsA expression or whether the reduction of PgsA expression by HSGN-94 is via a separate mechanism. This will be the focus of a future study.

Figure 8.

Proposed mechanism for HSGN-94 inhibition of LTA biosynthesis. We hypothesize that HSGN-94 inhibits LTA biosynthesis in a dual-mechanistic way.

In addition to LTA inhibition, the global proteomics data (Tables 1 and 3) also showed that treatment of S. aureus with HSGN-94 downregulated proteins involved in translation (mtaB), transcription (SarR and GntR), and nucleotide metabolism (GloB, PyrF, and CarB). Furthermore, we performed GO function analysis using Metascape^™ software of the 423 significant proteins (p < 0.05) and identified that nucleotide metabolic and small molecules catabolic processes, as well as precursor metabolites and energy were the most significantly regulated biological processes identified (−log10(p) >10, see Figure S4). Likewise, the proteomics data using affinity HSGN-probe (Table S4) indicated that HSGN-94 interacted with proteins involved in protein synthesis (miaB, rplD, PheT, rplJ, and trmFO), DNA/RNA synthesis (SigA, rpiA, Xpt, RecA, adk, and guaA), as well as cell-wall synthesis (IsaA). Therefore, to further evaluate this, we performed a macromolecular biosynthesis inhibition assay to analyze the integration of radiolabeled precursors into the biosynthesis of macromolecules (DNA, RNA, cell wall, and proteins). As expected from the data outlined in Tables 1, 3, and S4, HSGN-94 exhibited similar or superior inhibition of the biosynthesis of each macromolecule tested as compared to FDA approved antibiotics (Figure S5A-D). It thus appears that the potent antimicrobial activity of HSGN-94 is derived from LTA biosynthesis inhibition and the inhibition of other essential processes in bacteria. This multi-pronged inhibition of essential processes in bacteria explains why attempts to generate resistant clones towards HSGN-94 failed. It has emerged that successful antibiotics used in the clinic, while developed against single targets, are successful because they indeed target other pathways⁵⁵. For example, daptomycin, which has long been categorized as mainly acting via depolarization of bacterial membrane has now been found to also target cell wall biosynthesis via a mechanism that involves complex formation with undecaprenyl-coupled intermediates and membrane lipids⁵⁶. Recently, it was also reported that tetracyclines, long thought of as mainly acting via ribosome inhibition, also act via bacterial membrane targeting⁵⁷.

In vivo efficacy of HSGN-94 in a MRSA murine skin infection:

Based on its potent antibacterial activity, interesting mechanism of action, and the fact that HSGN-94 was not toxic to HaCat cells at concentrations up to 64 μg/mL, the compound was evaluated for its in vivo efficacy in a MRSA murine skin infection model. MRSA-infected wounds were treated as either clindamycin I.P. (25 mg/kg once daily), 2% HSGN-94, 2% mupirocin, or the vehicle alone (petroleum jelly) for five days. twice daily with either 2% HSGN-94, 2% mupirocin, or the vehicle alone (petroleum jelly). Excitingly, HSGN-94 (93.8% reduction) performed similarly to FDA-approved antibiotics clindamycin (93.4% reduction) and mupirocin (98.8% reduction) in lessening the quantity of MRSA in the infected wounds of mice after 5 days (Figure 9).

Figure 9: Reduction of MRSA USA300 in infected wounds of mice after 5 days.

The data are presented as average percent reduction of MRSA CFU/mL in murine skin wounds. A one-way ANOVA with post-hoc Dunnet’s multiple assessments discovered no statistical variation amongst mice treated with mupirocin or clindamycin and mice treated with HSGN-94 (2%).

HSGN-94 reduces pro-inflammatory cytokines:

The severity of S. aureus skin infections is propelled by the overabundance of host pro-inflammatory cytokines⁵⁸. Several reports have shown that in S. aureus infected wounds, LTA contributes to the increased development of inflammation and skin barrier defects^59–62. Since HSGN-94 is a potent LTA inhibitor and effectively reduced MRSA USA300 in infected wounds, we proceeded to examine its effect on pro-inflammatory cytokine expression in the treated wounds. . As depicted from Figure 10A–C, HSGN-94 reduced the levels of the pro-inflammatory cytokines, monocyte chemo attractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1β) in MRSA USA300 skin lesions. Additionally, the reduction of the levels of IL-1β and MCP-1 in response to HSGN-94 was was higher than that generated by mupirocin and clindamycin treatment (Figure 10A and 10B). It was reported that persistent inflammation because of inflammatory cytokines like MCP-1 and TNF-α, significantly impedes recovery in chronic wounds⁶³. It is exciting that HSGN-94 treatment resulted in the reduction of pro-inflammator cytokines, which could result in acceleration of wound healing.

Figure 10.

HSGN-94 Reduces Pro-Inflammatory Cytokines in MRSA Infected Wounds. For A to C, samples were pooled and average values are shown. (A) Level (pg/mL) of IL-1β in MRSA infected wound treated with vehicle, mupirocin, clidamycin, or HSGN-94. (B) Level (pg/mL) of MCP-1 in MRSA infected wound treated with vehicle, mupirocin, clidamycin, or HSGN-94. (C) Level (pg/mL) of TNF-α in MRSA infected wound treated with vehicle, mupirocin, clidamycin, or HSGN-94. (D) MRSA infected wound treated with vehicle with diffuse necrosis, chronic inflammation, edema and bacterial colonization present. (E) MRSA infected wound treated with mupirocin with moderate necrosis, chronic inflammation, and hemorrahge. (F) MRSA infected wound treated with clidamycin demonstrated epithelial hyperplasia on wound periphery, with moderate chronic inflammation and necrosis. (G) Histopathology of MRSA infected wound treated with HSGN-94 demonstrated resolution of inflammation within the deep dermis and subcutis.

To further evaluate HSGN-94’s effect on pro-inflammatory cytokines, we performed histopathology (Figure 10D–G). It is evident that MRSA infected wounds treated with mupirocin (Figure 10E), clidamycin (Figure 10F), or HSGN-94 (Figure 10G) demonstrate evidence of necrosis, hemorrhage, and a chronic inflammatory response. However, the response is less pronounced than the than vehicle treated group (Figure 10D). However, HSGN-94 showed resolution of inflammation and necrosis which was similar to clidamycin treatment but was much superior than mupriocin treatment (see Figure 10E–G). MRSA infected wounds treated with HSGN-94 show reduced levels of pro-inflammatory cytokines, which is superior to that of wounds treated with clindamycin or mupirocin and may be due to HSGN-94’s ability to inhibit LTA biosynthesis.

Conclusion:

In conclusion, using a panoply of techniques, including global proteomics, activity-based protein profiling, lipid analysis, and MRM profiling experiments, we have been able to propose how HSGN-94 inhibits LTA biosynthesis. We propose that HSGN-94 inhibits LTA in two ways: direct binding to PgcA which causes inhibition of Glc₂-DAG; and downregulation of PgsA, which leads to a reduction in PG synthesis. Excitingly, HSGN-94 showed high efficacy in diminishing the burden of MRSA in a murine skin infection model and also reduced the pro-inflammatory cytokines in MRSA infected wounds. Antimicrobial resistance is a growing threat and many groups have disclosed new chemical scaffolds that inhibit bacterial growth^64–74. Detailed mechanistic work to uncover how each of these unique compounds killing bacteria would likely reveal many novel tactics to tackle this global health challenge.

Experiemental Section:

Chemistry:

All reagents and solvents were purchased from commercial sources and utilized without purification. The ¹H, ¹³C, and ¹⁹F NMR spectra were obtained in DMSO-d6, chloroform-d, or methanol-d₄ as solvent using a 500 MHz or 800 MHz spectrometer with tetramethylsilane as the internal standard. ¹H NMR spectra data are reported as chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). High resolution mass spectra (HRMS) were taken using electron spray ionization (ESI) and a TOF mass analyzer. Characterization of compounds was done using ¹H NMR, ¹³C NMR, ¹⁹F NMR, and HRMS data. The purity of compounds was determined to be ≥95% by measuring the absorbance at 280 nm with high performance liquid chromatography (HPLC). HPLC spectra were recorded on an Agilent 1260 Infinity system using a ZORBAX SB-C18 column.

Synthetic Procedures:

General Procedure I: Synthesis of Sulfonamide-Containing Intermediates S1-S10, E1-E2, I5, I6, I8, and I10

To a solution of chlorosulfonyl-containing intermediate (1 eq) in methanol (25 mL) was added amine (3 eq). This mixture was stirred overnight at room temperature. Next, the mixture was concentrated under reduced pressure, diluted with ethyl acetate (20 mL), washed twice with water (10 mL), once with brine (10 mL), dried over Na₂SO₄, and concentrated under reduced pressure. The crude mixture was then purified via column chromatography using hexanes: ethyl acetate (80:20) as solvent system to give pure product.

General Procedure II: Synthesis of Aromatic 1,3,4-oxadiazol-2-amines A1:

The synthesis of A1 was performed using a literature-reported procedure⁷⁵. Obtained ¹H, ¹³C, and ¹⁹F spectra were in agreement with literature-reported data.

General Procedure III: Sandmeyer Reaction for the Synthesis of I1, I3, and I4

Thionyl chloride (5.5 eq) was added dropwise to water (15 mL) at 0°C and then stirred for 18 hours at room temperature. CuCl₂ (0.05 eq) was added at 0°C and the mixture was stirred for 15 minutes. In a separate flask, a solution of NaNO₂ (1.5 eq) in water (5 mL) was added to a stirred solution of 4-amino-3-nitrobenzonitrile (1 eq), 4-amino3-methoxybenzonitrile (1 eq), or 5-aminonicotinic acid (1 eq) in concentrated HCl (5 mL) at 0°C, over 15 minutes. The diazonium salt solution was added dropwise to the thionyl chloride/CuCl₂ solution at 0°C and stirred for 1 hour. Over this time, a precipitate formed which was collected via vacuum filtration and washed with water (10 mL) which gave the sulfonylchloride intermediates I1, I3, or I4. These intermediates were used in the next step without purification or characterization.

General Procedure IV: Hydrolysis of Benzonitriles I5-I7 to Benzoic Acids S13, S14, and S15

To a mixture of benzonitriles I5-I7 in ethanol (10 mL) was added sodium hydroxide (2M in H₂O) (16.6 mL) and the solution was refluxed at 100°C overnight. After, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was dissolved in H₂O and acidified to pH = 1 with 6 M HCl to give an off-white precipitate which was collected via vacuum filtration to give desired product which was continued without further purification or characterization.

General Procedure V: Hydrolysis of Esters E1 and E2 to Carboxylic Acids S11 and S12

To a solution of methyl ester E1 or E2 (1 eq) in MeOH: H₂O (2:1) was added LiOH (22 eq) and the solution was stirred at room temperature for 12 hours. After, the mixture was concentrated under reduced pressure and the crude product was dissolved in H₂O and acidified to pH = 1 with 6 M HCl to afford the carboxylic acid intermediate as an off-white solid which was collected via vacuum filtration.

General Procedure VI: Reduction of Nitro for the Synthesis of 15 and I9:

To a 50 mL round-bottom flask charged with nitro containing compound 13 (1 eq) or intermediate I8 (1 eq) was dissolved in DMF: H₂O (9:1). Na₂S₂O₄ (3.5 eq) was added, and the reaction was run at 90°C for 12 hours. After, the reaction mixture was concentrated under reduced pressure and diluted with ethyl acetate (20 mL). The organic layer was washed twice with H₂O (10 mL) and once with brine (10 mL). The organic layer was separated, dried over Na₂SO₄, and concentrated under reduced pressure to give a crude mixture which was purified via column chromatography (hexanes/ethyl acetate 70:30).

General Procedure VII: Synthesis of Alkyl containing 1,3,4-oxadiazol-2-amines A2 and A3:

The synthesis of A2 and A3 was performed using a literature-reported procedure⁷⁶. Obtained ¹H, and ¹³C spectra were in agreement with literature-reported data.

General Procedure VIII: Amide Coupling for the Synthesis of Compounds 1–14, 16, 18, and I11:

To a round-bottomed flask was added benzoic acid (1 eq), amine (1 eq), BOP reagent (2.7 eq), and DIPEA (1.5 mL) in DMF solvent (5 mL) was stirred at room temperature for 24 h. After completion, the reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified by flash column chromatography (hexanes/ethyl acetate 90:10 to 70:30) to give the desired product.

General Procedure IX: Synthesis of Compounds 19 and 20:

To a round-bottom flask with acyl chloride (1.4 eq) and 1,3,4-oxadiazol-2-amine (1 eq) in 1,4, -dioxane (5 mL) was added N-methylimidazole (5.5 eq). The mixture was stirred at 90°C for 2 hours and then concentrated under reduced pressure. The crude mixture was diluted in ethyl acetate (20 mL), washed twice with water (10 mL), once with brine (10 mL), dried over Na₂SO₄, and concentrated under reduced pressure. Column chromatography (hexanes/ethyl acetate 70:30) gave desired product.

Synthesis of Intermediate I2:

To a round-bottomed flask containing thiophene-2-carboxylic acid was slowly added chlorosulfonic acid (10 mL) at 0°C. After addition, the reaction was stirred at room temperature overnight. Then, the mixture was slowly poured over ice and extracted with ethyl acetate (3 × 30 mL). The combined organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give chlorosulfonyl containing intermediate I2 which was proceeded to the next step without further purification or characterization.

Synthesis of Intermediate I7:

To a round-bottom flask containing I6 (1 eq) in anhydrous DCM (10 mL) under argon gas was slowly added BBr₃ (5 eq) at −78°C. The mixture was stirred at −78°C for 1 hour and then warmed to room temperature to continue stirring for 12 hours. After, the reaction was cooled to 0°C and quenched slowly with iced water (10 mL). Next, the two layers were separated, and the water layer extracted twice with DCM (10 mL). The organic layers were combined and washed once with NaHCO₃ (10 mL), once with brine (10 mL), dried over Na₂SO₄, and concentrated under reduced pressure to give a crude mixture which was purified via column chromatography (hexanes: ethyl acetate (95:5) to give intermediate I7 as an off-white solid.

Synthesis of Analog 17:

To a solution of HSGN-94 (1 eq) in dry DMF (5 mL), sodium hydride, NaH (3 eq), was added, and the reaction mixture was allowed to stir for 30 min at room temperature. Then, methyl iodide (2 eq) was added dropwise, and the mixture was stirred for 2 h at room temperature. The reaction mixture was concentrated under reduced pressure and the residue was treated with aqueous NH₄OH (30% in water, 10 mL). The crude product was extracted with Et₂O (3 × 15 mL) and purified by silica gel column chromatography using hexanes: ethyl acetate (70:30) to give desired product.

Synthesis of HSGN-Probe:

To a solution of I11 (14 mg, 31.1 μmol) and biotin-PEG₃-N₃ (20 mg, 45 μmol, 1.5 eq) in DMF (2 mL) was added a mixture of CuSO₄·5H₂O (3 mg) and sodium ascorbate (3 mg) in water (0.2 mL). The solution was allowed to stir at 60°C for 12 hours. The solvents were evaporated, and the product was purified by column chromatography using DCM/MeOH (90/10) as eluent to obtain HSGN-Probe (77% yield) as a clear oil.

Characterization Data:

4-(Piperidin-1-ylsulfonyl)benzoic acid (S1):

Off-white solid (190 mg, 71% yield). ¹H NMR (500 MHz, DMSO-d6) δ 8.14 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 2.90 (d, J = 5.4 Hz, 4H), 1.52 (t, J = 5.7 Hz, 4H), 1.35 (d, J = 3.7 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d6) δ 166.7, 139.8, 135.1, 130.6, 128.1, 47.0, 25.1, 23.2. HRMS (ESI) m/z calcd for C₁₂H₁₆NO₄S [M + H]⁺ 270.0794, found 270.0793.

4-(Pyrrolidin-1-ylsulfonyl)benzoic acid (S2):

Off-white solid (154 mg, 44% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.15 – 8.14 (m, 2H), 7.91 – 7.90 (m, 2H), 3.19 – 3.18 (m, 4H), 1.68 (q, J = 3.7 Hz, 4H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 140.8, 135.1, 130.5, 127.8, 48.2, 25.1. HRMS (ESI) m/z calcd for C₁₁H₁₄NO₄S [M + H]⁺ 256.0644, found 256.0646.

4-(N-Cyclopropylsulfamoyl)benzoic acid (S3):

Off-white solid (166 mg, 51% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.14 (d, J = 8.0 Hz, 2H), 7.93 – 7.92 (m, 3H), 2.19 – 2.18 (m, 1H), 0.50 (dd, J = 7.0, 2.1 Hz, 2H), 0.40 (t, J = 3.1 Hz, 2H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.6, 144.6, 134.7, 130.3, 127.4, 24.5, 5.5. HRMS (ESI) m/z calcd for C₁₀H₁₂NO₄S [M + H]⁺ 242.0487, found 242.0484.

4-(N,N-Dimethylsulfamoyl)benzoic acid (S4):

Off-white solid (177 mg, 57% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.16 (dd, J = 8.4, 1.8 Hz, 2H), 7.86 (dd, J = 8.4, 1.7 Hz, 2H), 2.66 (s, 6H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 139.4, 135.2, 130.5, 128.0, 37.8. HRMS (ESI) m/z calcd for C₉H₁₂NO₄S [M + H]⁺ 230.0487, found 230.0488.

4-((2-Azabicyclo[2.2.1]heptan-2-yl)sulfonyl)benzoic acid (S5):

Off-white solid (210 mg, 55% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.14 – 8.13 (m, 2H), 7.92 – 7.91 (m, 2H), 4.14 (s, 1H), 3.06 – 3.04 (m, 1H), 2.99 (d, J = 9.0 Hz, 1H), 2.45 (s, 1H), 1.56 – 1.54 (m, 3H), 1.32 – 1.30 (m, 1H), 1.20 (d, J = 9.9 Hz, 1H), 0.84 (d, J = 10.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 142.5, 134.9, 130.5, 127.7, 60.3, 54.7, 37.4, 36.6, 31.2, 27.1. HRMS (ESI) m/z calcd for C₁₃H₁₆NO₄S [M + H]⁺ 282.0800, found 282.0801.

4-((4-Methylpiperidin-1-yl)sulfonyl)benzoic acid (S6):

Off-white solid (235 mg, 61% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.15 – 8.14 (m, 2H), 7.85 – 7.84 (m, 2H), 3.64 (d, J = 12.1 Hz, 2H), 2.35 (td, J = 12.1, 2.7 Hz, 2H), 1.66 – 1.64 (m, 2H), 1.36 – 1.32 (m, 1H), 1.15 (qd, J = 12.2, 4.0 Hz, 2H), 0.86 (d, J = 6.5 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 140.4, 135.2, 130.5, 127.9, 46.3, 33.3, 29.6, 21.5. HRMS (ESI) m/z calcd for C₁₃H₁₈NO₄S [M + H]⁺ 284.0957, found 284.0956.

4-((3-Methylpiperidin-1-yl)sulfonyl)benzoic acid (S7):

Off-white solid (227 mg, 59% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.15 – 8.14 (m, 2H), 7.85 – 7.84 (m, 2H), 3.52 (ddd, J = 16.2, 11.9, 6.1 Hz, 2H), 2.36 (td, J = 11.5, 3.2 Hz, 1H), 2.05 – 2.03 (m, 1H), 1.68 – 1.60 (m, 3H), 1.47 (dt, J = 12.9, 3.9 Hz, 1H), 0.90 – 0.87 (m, 1H), 0.84 (d, J = 6.4 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 140.4, 135.1, 130.5, 127.9, 52.9, 46.4, 31.6, 30.5, 24.5, 18.9. HRMS (ESI) m/z calcd for C₁₃H₁₈NO₄S [M + H]⁺ 284.0957, found 284.0959.

4-((2-Methylpiperidin-1-yl)sulfonyl)benzoic acid (S8):

Off-white solid (247 mg, 64% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.12 – 8.11 (m, 2H), 7.91 – 7.90 (m, 2H), 4.13 (d, J = 4.8 Hz, 1H), 3.64 (dt, J = 13.2, 3.2 Hz, 1H), 3.02 (td, J = 13.1, 2.7 Hz, 1H), 1.55 – 1.49 (m, 2H), 1.43 – 1.41 (m, 3H), 1.22 (qt, J = 12.5, 4.2 Hz, 1H), 1.03 (d, J = 7.0 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.5, 145.1, 134.7, 130.6, 127.2, 48.8, 40.5, 30.2, 25.1, 18.0, 15.8. HRMS (ESI) m/z calcd for C₁₃H₁₈NO₄S [M + H]⁺ 284.0957, found 284.0957.

5-(3,5-Dimethylpiperidin-1-yl)sulfonyl)nicotinic acid (S9):

Off-white solid (173 mg, 53% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 9.29 (s, 1H), 9.11 (s, 1H), 8.42 (d, J = 1.8 Hz, 1H), 3.69 – 3.67 (m, 2H), 1.93 (t, J = 11.3 Hz, 2H), 1.66 (tdd, J = 17.8, 15.2, 7.5, 3.8 Hz, 3H), 0.84 (d, J = 6.5 Hz, 6H), 0.57 (q, J = 12.1 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.3, 153.9, 151.1, 135.7, 133.8, 127.9, 52.3, 40.9, 31.0, 18.9. HRMS (ESI) m/z calcd for C₁₃H₁₉N₂O₄S [M + H]⁺ 299.1066, found 299.1067.

4-(3,5-Dimethylpiperidin-1-yl)sulfonyl)thiophene-2-carboxylic acid (S10):

Off-white solid (220 mg, 66% yield). ¹H NMR (500 MHz, DMSO-d6) δ = 8.45 (d, J = 1.6, 1H), 7.72 (d, J = 1.6, 1H), 3.60 – 3.57 (m, 2H), 1.78 (t, J = 11.2, 2H), 1.64 – 1.61 (m, 3H), 0.82 (d, J = 6.4, 6H), 0.57 – 0.49 (m, 1H). ¹³C NMR (126 MHz, DMSO-d6) δ = 162.4, 137.7, 137.6, 136.7, 130.7, 52.5, 40.9, 31.0, 19.2. HRMS (ESI) m/z calcd for C₁₂H₁₈NO₄S₂ [M + H]⁺ 304.0677, found 304.0676.

3-((3,5-Dimethylpiperidin-1-yl)sulfonyl)thiophene-2-carboxylic acid (S11):

Off-white solid (121 mg, 59% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 7.85 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 5.2 Hz, 1H), 3.77 – 3.75 (m, 2H), 2.14 (t, J = 11.8 Hz, 2H), 1.69 (d, J = 12.9 Hz, 1H), 1.58 – 1.55 (m, 2H), 0.82 (d, J = 6.7 Hz, 6H), 0.64 (q, J = 12.2 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 161.4, 139.7, 136.3, 130.3, 130.2, 130.1, 130.0, 52.5, 41.3, 31.0, 19.0. HRMS (ESI) m/z calcd for C₁₂H₁₈NO₄S₂ [M + H]⁺ 304.0677, found 304.0678.

5-((3,5-Dimethylpiperidin-1-yl)sulfonyl)furan-2-carboxylic acid (S12):

Off-white solid (171 mg, 60% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 7.32 – 7.31 (m, 1H), 7.23 (d, J = 3.6 Hz, 1H), 3.66 – 3.64 (m, 2H), 2.16 (t, J = 11.7 Hz, 2H), 1.70 – 1.68 (m, 1H), 1.61 – 1.59 (m, 2H), 0.85 (d, J = 6.8 Hz, 6H), 0.65 (q, J = 12.3 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 158.8, 149.8, 148.1, 118.0, 117.6, 52.2, 40.9, 30.9, 18.9. HRMS (ESI) m/z calcd for C₁₂H₁₈NO₅S [M + H]⁺ 288.0906, found 288.0907.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-3-nitrobenzonitrile (I5):

Yellow solid (280 mg, 61% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.26 (d, J = 2.1 Hz, 1H), 7.82 (dd, J = 8.9, 2.2 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 3.25 – 3.21 (m, 2H), 2.54 – 2.49 (m, 2H), 1.77 (d, J = 12.1 Hz, 1H), 1.69 – 1.64 (m, 2H), 0.83 (d, J = 6.5 Hz, 6H), 0.78 (q, J = 12.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 147.8, 138.7, 136.7, 131.8, 121.6, 118.4, 99.6, 57.3, 41.4, 31.1, 19.1. HRMS (ESI) m/z calcd for C₁₄H₁₈N₃O₄S [M + H]⁺ 324.1018, found 324.1015.

4-((3,5-dimethylpiperidin-1-yl)sulfonyl)-3-methoxybenzonitrile (I6):

Off-white solid (273 mg, 51% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 7.87 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 1.5 Hz, 1H), 7.55 (dd, J = 8.1, 1.4 Hz, 1H), 3.93 (s, 3H), 3.61 (dd, J = 12.1, 4.0 Hz, 2H), 2.13 – 2.08 (m, 2H), 1.67 (ddt, J = 13.2, 4.1, 2.2 Hz, 1H), 1.55 (dq, J = 11.0, 3.6 Hz, 2H), 0.80 (d, J = 6.6 Hz, 6H), 0.62 (q, J = 12.0 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.0, 131.8, 131.4, 124.7, 118.1, 117.4, 117.0, 57.2, 52.3, 41.3, 31.4, 19.1. HRMS (ESI) m/z calcd for C₁₅H₂₁N₂O₃S [M + H]⁺ 309.1273, found 309.1270.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-3-hydroxybenzonitrile (I7):

Off-white solid (158 mg, 72% yield). ¹H NMR (500 MHz, Chloroform-d) δ 9.07 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 1.6 Hz, 1H), 7.26 (dd, J = 8.2, 1.6 Hz, 1H), 3.71 – 3.68 (m, 2H), 1.90 – 1.85 (m, 2H), 1.75 – 1.72 (m, 3H), 0.86 (d, J = 6.5 Hz, 6H), 0.58 – 0.50 (m, 1H). ¹³C NMR (126 MHz, Chloroform-d) δ 155.4, 129.4, 129.4, 124.3, 123.2, 123.2, 122.8, 122.7, 118.2, 117.0, 52.4, 41.0, 30.9, 18.9. HRMS (ESI) m/z calcd for C₁₄H₁₉N₂O₃S [M + H]⁺ 295.1116, found 295.1116.

3,5-Dimethyl-1-((4-nitrophenyl)sulfonyl)piperidine (I8):

Yellow solid (214 mg, 53% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.42 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 3.67 – 3.65 (m, 2H), 1.90 (t, J = 11.4 Hz, 2H), 1.68 – 1.61 (m, 3H), 0.83 (d, J = 6.6 Hz, 6H), 0.57 (q, J = 12.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 150.4, 142.6, 129.2, 129.1, 125.0, 124.9, 52.4, 41.0, 30.9, 18.9. HRMS (ESI) m/z calcd for C₁₃H₁₉N₂O₄S [M + H]⁺ 299.1066, found 299.1065.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)aniline (I9):

Off-white solid (115 mg, 64% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 7.37 (d, J = 9.1 Hz, 2H), 6.67 (d, J = 8.3 Hz, 2H), 5.85 (s, 2H), 3.54 (dd, J = 11.1, 3.3 Hz, 2H), 1.67 – 1.60 (m, 5H), 0.81 (d, J = 6.1 Hz, 6H), 0.49 (q, J = 11.9 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 153.3, 129.6, 129.5, 121.6, 113.3, 113.2, 52.8, 41.3, 30.8, 19.2. HRMS (ESI) m/z calcd for C₁₃H₂₁N₂O₂S [M + H]⁺ 269.1324, found 269.1326.

4-(N-(Prop-2-yn-1-yl)sulfamoyl)benzoic acid (I10):

Off-white solid (173 mg, 63% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.19 (t, J = 6.0 Hz, 1H), 8.11 – 8.10 (m, 2H), 7.93 – 7.92 (m, 2H), 3.76 (d, J = 3.6 Hz, 2H), 2.94 (s, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.6, 144.8, 134.7, 130.3, 127.3, 79.6, 75.0, 32.3. HRMS (ESI) m/z calcd for C₁₀H₁₀NO₄S [M + H]⁺ 240.0331, found 240.0328.

4-(N-(Prop-2-yn-1-yl)sulfamoyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (I11):

Off-white solid (45 mg, 24% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.37 (t, J = 5.9 Hz, 1H), 8.21 – 8.16 (m, 4H), 7.99 – 7.96 (m, 4H), 3.77 (dd, J = 5.9, 2.5 Hz, 2H), 3.03 (t, J = 2.5 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 164.5, 160.3, 158.7, 144.8, 136.1, 132.0 (q, J = 34.2 Hz), 129.5, 127.5, 127.4, 127.3, 127.1, 126.9, 126.8, 126.6, 126.2, 124.8 (q, J = 273.4 Hz), 79.6, 75.0, 32.4. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₁₉H₁₄F₃N₄O₄S [M + H]⁺ 451.0688, found 451.0690.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (HSGN-94):

The synthesis and characterization of HSGN-94 was demonstrated in our previous report²⁷. For in vivo analysis, the compound was scaled up. Purity by HPLC was found to be 98%.

4-(Piperidin-1-ylsulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (1):

Off-white solid (53 mg, 30% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.26 – 8.25 (m, 2H), 8.19 – 8.18 (m, 2H), 7.98 – 7.97 (m, 2H), 7.91 – 7.90 (m, 2H), 3.01 (t, J = 5.5 Hz, 4H), 1.57 (q, J = 5.8 Hz, 4H), 1.41 (t, J = 5.9 Hz, 2H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.3, 159.9, 159.0, 140.3, 137.0, 132.0 (q, J = 32.2 Hz), 129.7, 127.9, 127.5, 127.3, 126.7, 124.8 (q, J = 271.4 Hz), 46.9, 25.1, 23.3. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₁H₂₀F₃N₄O₄S [M + H]⁺ 481.1157, found 481.1155. Purity by HPLC was found to be 98%.

4-(Pyrrolidin-1-ylsulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (2):

Off-white solid (32 mg, 18% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.33 – 8.32 (m, 2H), 8.14 – 8.12 (m, 2H), 7.92 – 7.88 (m, 4H), 3.20 – 3.18 (m, 4H), 1.68 (d, J = 6.5 Hz, 4H). ¹³C NMR (201 MHz, DMSO-d₆) δ 171.4, 168.7, 156.8, 141.8, 139.4, 131.4 (q, J = 32.2 Hz), 129.8, 128.2, 127.5, 126.9, 126.6, 124.9 (q, J = 271.4 Hz), 48.2, 25.1. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₀H₁₈F₃N₄O₄S [M + H]⁺ 467.1001, found 467.1000. Purity by HPLC was found to be 99%.

4-(N-cyclopropylsulfamoyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (3):

Off-white solid (41 mg, 22% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.24 – 8.23 (m, 2H), 8.19 – 8.18 (m, 2H), 7.99 – 7.97 (m, 4H), 2.23 (tq, J = 6.8, 3.3 Hz, 1H), 0.53 (dd, J = 7.1, 2.2 Hz, 2H), 0.42 – 0.41 (m, 2H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.2, 160.0, 158.9, 144.6, 136.5, 132.0 (q, J = 32.2 Hz), 129.6, 127.5, 127.3, 126.8, 126.2, 124.8 (q, J = 273.4 Hz), 24.6, 5.5. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₁₉H₁₆F₃N₄O₄S [M + H]⁺ 453.0844, found 453.0844. Purity by HPLC was found to be 95%

4-(N,N-Dimethylsulfamoyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (4):

Off-white solid (49 mg, 26% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.27 – 8.26 (m, 2H), 8.19 – 8.18 (m, 2H), 7.98 – 7.97 (m, 2H), 7.94 – 7.93 (m, 2H), 2.70 (s, 6H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.3, 160.0, 158.9, 139.4, 137.0, 132.0 (q, J = 32.2 Hz), 129.7, 128.0, 127.5, 127.3, 126.8, 124.8 (q, J = 271.4 Hz), 37.9. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₁₈H₁₆F₃N₄O₄S [M + H]⁺ 441.0844, found 441.0847. Purity by HPLC was found to be 99%.

4-((2-Azabicyclo[2.2.1]heptan-2-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (5):

Off-white solid (26 mg, 15% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.24 – 8.23 (m, 2H), 8.19 – 8.18 (m, 2H), 7.99 – 7.97 (m, 4H), 4.19 (s, 1H), 3.10 (dd, J = 9.0, 2.8 Hz, 1H), 3.03 (d, J = 9.1 Hz, 1H), 2.49 (s, 1H), 1.61 – 1.57 (m, 3H), 1.35 – 1.34 (m, 1H), 1.23 (d, J = 9.9 Hz, 1H), 0.89 (dt, J = 9.9, 2.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.3, 160.0, 159.0, 142.4, 136.8, 132.0 (q, J = 32.2 Hz), 129.7, 127.7, 127.5, 127.3, 126.8, 124.8 (q, J = 271.4 Hz), 60.3, 54.7, 37.4, 36.6, 31.3, 27.1. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₂H₂₀F₃N₄O₄S [M + H]⁺ 493.1157, found 493.1156. Purity by HPLC was found to be 98%.

4-((4-Methylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (6):

Off-white solid (40 mg, 23% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.26 – 8.25 (m, 2H), 8.18 – 8.17 (m, 2H), 7.98 – 7.97 (m, 2H), 7.92 – 7.91 (m, 2H), 3.67 (d, J = 11.9 Hz, 2H), 2.38 – 2.35 (m, 2H), 1.67 – 1.65 (m, 2H), 1.36 – 1.34 (m, 1H), 1.16 (qd, J = 12.1, 3.9 Hz, 2H), 0.87 (d, J = 6.6 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.2, 159.9, 159.0, 140.4, 137.0, 132.0 (q, J = 32.2 Hz), 129.7, 127.9, 127.5, 127.3, 126.8, 124.8 (q, J = 273.4 Hz), 46.3, 33.3, 29.6, 21.5. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₂H₂₂F₃N₄O₄S [M+H]⁺ 495.1310, found 495.1306. Purity by HPLC was found to be 97%.

4-((3-Methylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (7):

Off-white solid (36 mg, 21% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.26 – 8.25 (m, 2H), 8.19 – 8.18 (m, 2H), 7.98 – 7.97 (m, 2H), 7.92 – 7.91 (m, 2H), 3.55 (ddd, J = 16.2, 11.9, 5.7 Hz, 2H), 2.40 (td, J = 11.6, 2.8 Hz, 1H), 2.09 – 2.06 (m, 1H), 1.70 (dt, J = 13.5, 3.6 Hz, 1H), 1.67–1.62 (m, 2H), 1.50 – 1.45 (m, 1H), 0.93 (dtd, J = 14.8, 11.5, 3.7 Hz, 1H), 0.88 (d, J = 6.4 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.2, 159.9, 159.1, 140.4, 137.1, 131.9 (q, J = 32.2 Hz), 129.7, 127.8, 127.6, 127.3, 126.8, 124.8 (q, J = 271.4 Hz), 53.0, 46.5, 31.7, 30.5, 24.5, 18.9. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₂H₂₂F₃N₄O₄S [M+H]⁺ 495.1310, found 495.1312. Purity by HPLC was found to be 98%

4-((2-Methylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (8):

Off-white solid (24 mg, 14% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.22 – 8.21 (m, 2H), 8.19 – 8.18 (m, 2H), 7.98 – 7.97 (m, 4H), 4.18 – 4.16 (m, 1H), 3.68 – 3.65 (m, 1H), 3.05 (td, J = 13.1, 2.7 Hz, 1H), 1.58 – 1.51 (m, 2H), 1.44 (dq, J = 9.2, 5.6, 5.0 Hz, 3H), 1.25 (dt, J = 12.8, 4.3 Hz, 1H), 1.06 (d, J = 6.9 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.2, 160.0, 159.0, 145.0, 136.6, 132.0 (q, J = 32.2 Hz), 129.8, 127.5, 127.3, 127.2, 126.8, 126.8, 124.8 (q, J = 273.4 Hz), 48.8, 30.3, 25.1, 18.0, 15.9. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₂H₂₂F₃N₄O₄S [M+H]⁺ 495.1310, found 495.1311. Purity by HPLC was found to be 99%.

5-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)nicotinamide (9):

Off-white solid (37 mg, 22% yield). ¹H NMR (800 MHz, DMSO-d₆) δ = 9.40 (d, J=2.1, 1H), 9.12 (d, J=2.1, 1H), 8.67 (s, 1H), 8.17 (d, J=8.1, 2H), 7.99 (d, J=8.2, 2H), 3.69 (dd, J=11.4, 3.8, 2H), 1.89 (t, J=11.3, 2H), 1.66 (d, J=10.8, 3H), 0.82 (d, J=6.4, 6H), 0.57 – 0.50 (m, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 164.1, 159.6, 159.0, 153.3, 150.9, 135.1, 133.7, 132.0 (q, J = 32.2 Hz), 129.8, 127.4, 127.3, 126.8, 124.8 (q, J = 273.4 Hz), 52.3, 41.0, 31.0, 19.0. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₂H₂₃F₃N₅O₄S [M+H]⁺ 510.1417, found 510.1415. Purity by HPLC was found to be 99%

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)thiophene-2-carboxamide (10):

Off-white solid (49 mg, 29% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.35 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 3.59 (dd, J = 11.2, 3.8 Hz, 2H), 1.85 (t, J = 11.2 Hz, 2H), 1.68 – 1.61 (m, 3H), 0.83 (d, J = 6.4 Hz, 6H), 0.64 – 0.44 (m, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 160.0, 159.6, 159.0, 141.0, 137.5, 131.9 (q, J = 32.2 Hz), 129.4, 127.6, 127.3, 126.8, 126.2, 124.8 (q, J = 271.4 Hz), 52.5, 41.1, 31.0, 19.1. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₁H₂₂F₃N₄O₄S₂ [M+H]⁺ 515.1029, found 515.1029. Purity by HPLC was found to be 96%.

3-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)thiophene-2-carboxamide (11):

Off-white solid (30 mg, 18% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.14 – 8.13 (m, 2H), 7.97 – 7.96 (m, 2H), 7.91 (d, J = 5.2 Hz, 1H), 7.38 (d, J = 5.2 Hz, 1H), 3.72 (dd, J = 12.2, 4.0 Hz, 2H), 2.08 (t, J = 11.7 Hz, 2H), 1.68 (d, J = 13.1 Hz, 1H), 1.61 – 1.58 (m, 2H), 0.84 (d, J = 6.6 Hz, 6H), 0.63 (q, J = 12.1 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 159.6, 158.3, 139.5, 136.7, 131.9 (q, J = 32.2 Hz), 129.5, 127.9, 127.5, 127.3, 126.8, 126.7, 124.8 (q, J = 273.4 Hz), 52.4, 41.2, 30.9, 19.0. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₁H₂₂F₃N₄O₄S₂ [M+H]⁺ 515.1029, found 515.1026. Purity by HPLC was found to be 96%.

5-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)furan-2-carboxamide (12):

Off-white solid (38 mg, 22% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.17 – 8.16 (m, 2H), 7.98 – 7.97 (m, 2H), 7.62 (d, J = 3.8 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 3.70 (dd, J = 12.0, 4.1 Hz, 2H), 2.22 (t, J = 11.7 Hz, 2H), 1.71 (d, J = 13.4 Hz, 1H), 1.64 – 1.60 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H), 0.66 (q, J = 12.3 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 159.5, 158.9, 156.3, 150.0, 149.5, 132.0 (q, J = 30.2 Hz), 127.5, 127.3, 126.8, 124.8 (q, J = 273.4 Hz), 117.9, 117.7, 117.3, 52.3, 40.9, 30.9, 18.9. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₁H₂₂F₃N₄O₅S [M+H]⁺ 499.1263, found 499.1262. Purity by HPLC was found to be 97%.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-3-nitro-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (13):

Yellow solid (42 mg, 26% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.17 – 8.16 (m, 2H), 8.14 – 8.12 (m, 1H), 7.96 – 7.95 (m, 2H), 7.36 (d, J = 8.8 Hz, 1H), 3.29 – 3.27 (m, 2H), 2.54 (t, J = 12.0 Hz, 2H), 1.81 (d, J = 13.0 Hz, 1H), 1.75 – 1.74 (m, 1H), 0.88 (d, J = 6.6 Hz, 6H), 0.78 (q, J = 12.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 163.4, 160.1, 159.0, 148.2, 139.1, 133.6, 131.9 (q, J = 32.2 Hz), 127.6, 127.4, 127.2, 126.8, 126.7, 124.8 (q, J = 271.4 Hz), 120.7, 57.7, 41.5, 31.0, 19.0. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₃H₂₃F₃N₅O₆S [M+H]⁺ 554.1321, found 554.1322. Purity by HPLC was found to be 95%.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-3-methoxy-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (14):

Off-white solid (31 mg, 19% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.17 – 8.16 (m, 2H), 7.99 – 7.98 (m, 2H), 7.90 – 7.88 (m, 1H), 7.81 (d, J = 1.6 Hz, 1H), 7.71 – 7.69 (m, 1H), 3.97 (s, 3H), 3.64 (dd, J = 12.1, 4.0 Hz, 2H), 2.13 – 2.07 (m, 2H), 1.68 (d, J = 13.0 Hz, 1H), 1.56 (ddt, J = 14.5, 11.3, 4.6 Hz, 2H), 0.81 (d, J = 6.6 Hz, 6H), 0.62 (q, J = 12.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 164.0, 158.7, 156.9, 137.9, 131.3 (q, J = 32.2 Hz), 127.4, 127.0, 125.3, 123.1 (q, J = 271.4 Hz), 120.7, 113.3, 56.8, 52.5, 41.4, 31.4, 19.1. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₄H₂₆F₃N₄O₅S [M+H]⁺ 539.1576, found 539.1577. Purity by HPLC was found to be 95%.

3-Amino-4-((3,5-dimethylpiperidin-1-yl)sulfonyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (15):

Off-white solid (34 mg, 73% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.18 – 8.17 (m, 2H), 7.96 – 7.95 (m, 2H), 7.37 (d, J = 2.2 Hz, 1H), 7.34 – 7.32 (m, 1H), 6.97 (d, J = 8.1 Hz, 1H), 3.13 (dd, J = 11.3, 3.5 Hz, 2H), 2.09 (t, J = 11.0 Hz, 2H), 1.84 – 1.78 (m, 3H), 0.89 (d, J = 6.8 Hz, 6H), 0.68 (q, J = 12.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.8, 160.3, 159.2, 143.6, 142.4, 131.8 (q, J = 32.2 Hz), 127.7, 127.5, 127.2, 126.7, 124.8 (q, J = 271.4 Hz), 119.1, 117.9, 114.6, 58.3, 42.3, 31.5, 19.5. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₃H₂₅F₃N₅O₄S [M+H]⁺ 524.1579, found 524.1579. Purity by HPLC was found to be 95%.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-3-hydroxy-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (16):

Off-white solid (38 mg, 23% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.16 – 8.15 (m, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.1 Hz, 1H), 7.81 – 7.69 (m, 1H), 7.57 – 7.54 (m, 2H), 3.67 – 3.63 (m, 2H), 2.12 – 2.06 (m, 2H), 1.67 (d, J = 13.0 Hz, 1H), 1.57 (q, J = 8.8, 7.7 Hz, 2H), 0.80 (d, J = 6.6 Hz, 6H), 0.62 – 0.54 (m, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.3, 160.2, 158.7, 155.8, 138.3, 132.1 (q, J = 32.2 Hz), 131.1, 128.5, 127.4, 126.7, 123.3 (q, J = 271.4 Hz), 118.6, 117.7, 52.5, 41.3, 31.1, 18.9. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.7 (s, 3F). HRMS (ESI) m/z calcd for C₂₃H₂₄F₃N₄O₅S [M+H]⁺ 525.1420, found 525.1419. Purity by HPLC was found to be 95%.

4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)-N-methyl-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (17):

Off-white solid (29 mg, 57% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.38 – 8.37 (m, 2H), 8.12 – 8.11 (m, 2H), 8.00 – 7.99 (m, 2H), 7.86 – 7.84 (m, 2H), 3.75 (s, 3H), 3.66 (dd, J = 11.3, 3.8 Hz, 2H), 1.82 (t, J = 11.4 Hz, 3H), 1.67 – 1.62 (m, 3H), 0.83 (d, J = 6.6 Hz, 6H), 0.55 (q, J = 12.1, 11.5 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 170.1, 156.0, 154.3, 140.9, 132.3 (q, J = 32.2 Hz), 130.3, 128.8, 128.0, 127.1, 126.9, 126.8, 124.7 (q, J = 271.4 Hz), 52.6, 41.1, 34.9, 30.9, 19.0. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −62.8 (s, 3F). HRMS (ESI) m/z calcd for C₂₄H₂₆F₃N₄O₄S [M+H]⁺ 523.1627, found 523.1629. Purity by HPLC was found to be 95%.

N-(4-((3,5-Dimethylpiperidin-1-yl)sulfonyl)phenyl)-5-phenyl-1,3,4-oxadiazole-2-carboxamide (18):

Off-white solid (90 mg, 39% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.14 – 8.13 (m, 2H), 8.10 – 8.08 (m, 2H), 7.79 – 7.78 (m, 2H), 7.72 – 7.70 (m, 1H), 7.70 – 7.66 (m, 2H), 3.64 – 3.62 (m, 2H), 1.78 (t, J = 11.4 Hz, 2H), 1.66 – 1.62 (m, 3H), 0.84 (d, J = 6.5 Hz, 6H), 0.55 – 0.51 (m, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 165.9, 158.8, 152.3, 141.9, 133.2, 132.4, 129.9, 128.8, 127.6, 123.1, 121.3, 52.7, 41.1, 30.9, 19.1. HRMS (ESI) m/z calcd for C₂₂H₂₅N₄O₄S [M+H]⁺ 441.1591, found 441.1592. Purity by HPLC was found to be 96%.

N-(5-cyclohexyl-1,3,4-oxadiazol-2-yl)-4-((3,5-dimethylpiperidin-1-yl)sulfonyl)benzamide (19):

Off-white solid (45 mg, 32% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.21 – 8.20 (m, 2H), 7.89 – 7.88 (m, 2H), 3.67 (dd, J = 11.6, 3.9 Hz, 2H), 2.95 (ddd, J = 10.8, 7.0, 3.8 Hz, 1H), 2.03 (dd, J = 13.2, 4.0 Hz, 2H), 1.83 (t, J = 11.4 Hz, 2H), 1.77 (dt, J = 13.3, 4.1 Hz, 2H), 1.74 – 1.64 (m, 4H), 1.63 (qd, J = 11.4, 3.5 Hz, 2H), 1.44 (tdd, J = 15.4, 11.8, 3.6 Hz, 2H), 1.32 – 1.27 (m, 1H), 0.83 (d, J = 6.5 Hz, 6H), 0.55 (q, J = 12.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 166.3, 165.7, 158.2, 140.3, 137.5, 129.7, 127.8, 52.6, 41.1, 34.6, 30.9, 29.7, 25.6, 24.9, 19.0. HRMS (ESI) m/z calcd for C₂₂H₃₁N₄O₄S [M+H]⁺ 447.2066, found 447.2062. Purity by HPLC was found to be 95%.

N-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-4-((3,5-dimethylpiperidin-1-yl)sulfonyl)benzamide (20):

Off-white solid (30 mg, 24% yield). ¹H NMR (800 MHz, DMSO-d₆) δ 8.20 – 8.19 (m, 2H), 7.89 – 7.88 (m, 2H), 3.67 – 3.65 (m, 2H), 2.21 – 2.18 (m, 1H), 1.84 (t, J = 11.3 Hz, 2H), 1.67 – 1.62 (m, 3H), 1.14 – 1.13 (m, 2H), 1.02 – 1.01 (m, 2H), 0.83 (dd, J = 6.6, 1.3 Hz, 6H), 0.54 (q, J = 12.0 Hz, 1H). ¹³C NMR (201 MHz, DMSO-d₆) δ 164.8, 157.8, 140.3, 137.2, 129.6, 128.7, 127.8, 52.6, 41.1, 30.9, 19.0, 7.8, 6.2. HRMS (ESI) m/z calcd for C₁₉H₂₅N₄O₄S [M+H]⁺ 405.1597, found 405.1596. Purity by HPLC was found to be 97%.

4-(N-((1-(13-Oxo-17-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl)sulfamoyl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)benzamide (HSGN-Probe):

Clear oil (21 mg, 77% yield). ¹H NMR (800 MHz, Methanol-d₄) δ 8.23 – 8.19 (m, 4H), 8.00 – 7.95 (m, 2H), 7.90 – 7.88 (m, 2H), 7.82 (s, 1H), 4.49 (d, J = 4.2 Hz, 2H), 4.30 (dd, J = 7.8, 4.3 Hz, 1H), 4.25 (s, 2H), 3.82 (t, J = 4.7 Hz, 2H), 3.60 – 3.59 (m, 8H), 3.53 (t, J = 5.4 Hz, 2H), 3.20 (dt, J = 9.5, 4.9 Hz, 1H), 2.92 – 2.89 (m, 1H), 2.70 – 2.67 (m, 1H), 2.20 – 2.18 (m, 2H), 1.73 – 1.57 (m, 5H), 1.55 – 1.38 (m, 3H), 1.30 – 1.25 (m, 2H). ¹³C NMR (201 MHz, Methanol-d₄) δ 174.7, 165.1, 164.6, 159.4, 158.8, 144.4, 143.6, 136.4, 132.8 (q, J = 34.2 Hz), 128.8, 126.9, 126.0, 124.4 (q, J = 271.4 Hz), 123.9, 70.1, 70.0, 69.9, 69.8, 69.1, 68.8, 61.9, 60.1, 55.5, 49.8, 39.6, 38.8, 37.7, 35.3, 28.3, 28.0, 25.4. ¹⁹F NMR (471 MHz, Methanol-d₄) δ −65.7 (s, 3F). HRMS (ESI) m/z calcd for C₃₇H₄₆F₃N₁₀O₉S₂ [M+H]⁺ 895.2843, found 895.2846. Purity by HPLC was found to be 91%.

Biological analysis

Bacterial strains, media, cell lines and reagents

Clinical isolates used in this study (Table S5) were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and the American Type Culture Collection (ATCC). Cation-adjusted Mueller Hinton broth (CAMHB), tryptic soy broth (TSB) and tryptic soy agar (TSA) were purchased from Becton, Dickinson and Company (Cockeysville, MD, USA). Human keratinocyte cell line (HaCaT) was obtained from AddexBio (San Diego, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Corning (Manassas, VA, USA). Linezolid and vancomycin were purchase from Chem-Impex International (Wood Dale, IL, USA). Compounds were synthesized from commercial sources in our laboratory and prepared in stock solutions in DMSO.

Determination of the MICs against clinically-important Gram-positive bacteria

MICs of 1,3,4-oxadiazol-2-yl benzamides were determined using the broth microdilution method as outlined previously⁷⁷. Briefly, a bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in CAMHB to achieve a bacterial concentration of about 5 × 10⁵ CFU/mL and seeded in 96-well plates. Streptococci, enterococci and Listeria were diluted in TSB. Serial dilutions of tested agents were incubated with the bacteria aerobically at 37° C for 18–20 hours (except for S. pneumoniae which was incubated in presence of 5% CO₂). MICs were determined as the lowest concentration of the each test agent that completely inhibited the bacterial growth as determined visually.

In vitro cytotoxicity analysis of HSGN-94 against human keratinocytes (HaCat) cells

Cytotoxicity assessment for HSGN-94 was determined as previously described^78–79. HSGN-94 was assayed (at concentrations of 16, 32, 64 and 128 μg/mL) against human keratinocyte cells (HaCat) to determine its potential toxic effect to mammalian skin cells in vitro. Briefly, cells were incubated with the compound (in sextuplicate) at 37 °C with 5% CO₂ for 2 hours. DMSO was included as a control to determine the baseline measure of the cytotoxic impact of the compound. MTS 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for three hours before the absorbance readings (at OD₄₉₀) were recorded.

Multi-step Resistance Selection:

To assess if MRSA USA300 could form resistance to HSGN-94, a multi-step serial passaging experiment was conducted for 65 days, as described previously^{25, 80–81}. Resistance was considered as a greater than four-fold increase in the MIC as compared to the initial MIC.

Proteomics Analysis:

HSGN-94 (0.25 μg/mL) or a corresponding quantity of DMSO was added to a culture of S. aureus ATCC 25923 at OD₆₀₀ 0.6. The samples were treated for 2 hours at 37°C and then normalized to OD₆₀₀ 1.1 using sterile saline. Next, samples were centrifuged and then washed with PBS (2×). Both LC/MS/MS preparation and acquisition of data were executed as previously described⁸². Protein extraction was peformed using a Barocycler NEP2320 (Pressure Biosciences, South Easton, MA) system in which samples were lysed in 100 mM ammonium bicarbonate at 5 °C for 90 cycles (each cycle with 50 seconds at 35 kpsi and 10 seconds at atmospheric pressure). Protein concentration was measured by Bicinchoninic Acid (BCA) assay, and 50 μg (equivalent volume) of lysate was precipitated using four volume of cold (−20°C) 100% acetone overnight. Protein pellets were obtained via centrifugation of samples and then re-suspended in 8M urea with 10 mM DTT followed by incubation for 45 minutes. Next, cysteine alkylation was performed in the dark at room tempeature for 45 minutes with 20 mM iodoacetamide followed by the addition of 5 mM DTT and incubation at 37 °C for 20 minutes. Dilution of alkylated and reduced samples was executed to lessen the urea concentrataion to 1.5 M followed by overnight digestion at 37°C via 1:25 (w/w) enzyme-protein ratio using Trypsin/Lys-C Mix (Promega, Madison, WI, USA). Peptide desaltation was then performed by passing the digested peptides through C18 silica micro spin columns (The Nest Group Inc., Southborough, MA, USA). Then, the BCA assay was used to determine concentration of peptides as described above. The peptide concentration was altered to 0.2 μg/μL, and ~1 μg (5 μL) was used as injection volume for LC-MS/MS. A reverse-phase HPLC-ESI-MS/MS system containing an UltiMate^™ 3000 RSLCnano system joined to a Q-Exactive (QE) High Field (HF) Hybrid Quadrupole-Orbitrap^™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA) and a Nano-spray Flex^™ ion source (Thermo Fisher Scientific) was used to aquire the data. A resolution of 15,000 at m/z 200 was used for MS/MS scan acquisition and a 30 second dyanmic exclusion time was set to circumvent indistinguishable peptides. Both the MaxQuant^83–85 and Perseus³⁵ softwares were utilized for data and bioinformatics analysis respectively, as previously described⁸². GO function analysis was performed using Metascape^™ software.

Total RNA isolation and RT-PCR:

HSGN-94 (0.25 μg/mL) or DMSO was added to a culture of S. aureus ATCC 25923 at OD₆₀₀ 0.6 in triplicates. The samples were incubated for 2 hours at 37°C and then centrifuged to obtain sample pellets. RT-PCR was executed using a BioRad CFX96^™ Touch Real-Time PCR detection system follwing a procedure that has been previously described⁸². The PCR primers used were either contructed with Primer-BLAST or acquied from literature (Table S6). 16S rRNA was used as reference for data normalization. Statistical significance was sought by means of student’s t-test.

Pull-Down Assay:

The pull down assay and proteomic analysis was executed using a procedure outlined previously⁸⁶. Breifly, 50 mL of an overnight culture of S. aureus ATCC 25923 was grown to OD₆₀₀ = 1. This culture was pelleted by centrifugation and resuspended in 1 mL 10 mM Tris·HCl (pH 7.5), 50 mM NaCl buffer containing EDTA-free complete protease inhibitor (Roche). These cells were combined with 0.1-mm glass beads and lysed twice for 45 seconds using a Fast-Prep machine (MP Biomedicals). Samples were centrifuged at 17,000 × g for 5 minutes followed by centrifugation at 100,000 × g for 1 hour in order to obtain protein extracts. 40 μL streptavidin dynabeads (Invitrogen) coupled with either 2.4 μM HSGN-Probe or 2.4 μM HSGN-Probe plus 50 μM HSGN-94 were incubated at room temperature for 30 minutes with 1.2 mg cytoplasmic proteins in sample buffer prepared with 1.5 mL 10% glycerol, 1 mM MgCl₂, 230 mM NaCl, 0.5 mM DTT, 5 mM Tris (pH 7.5), and 4 mM EDTA containing 50 μg/mL BSA. After incubation, samples were washed 4 × with the sample buffer lacking BSA and suspended in 50 μL protein sample buffer. Next, samples were heated to boiling for 5 min and then the beads were removed. 18 μL of sample were run on 12% (wt/vol) SDS/PAGE gels which were then stained using the SilverQuest kit (Invitrogen). This solution then underwent LC/MS/MS analysis as described above. Data aqusition was performed using MaxQuant and bioinformatic analysis was done using Perseus as mentioned above.

Membrane Lipid Extraction and TLC Analysis:

Exponentially growing S. aureus ATCC 25923 was treated with either DMSO or HSGN-94 (0.25 ×, 1× and 8× MIC) for 5 hours at 37°C. The samples were normalized to OD₆₀₀ 0.9 and then centrifuged at 10,000 rpm for 10 minutes. The cells were collected and washed twice with 0.1 M sodium acetate (pH 4.7). Next, lipids were extracted following the Bligh-Dyer method⁸⁷ and lipids were isolated and dried over nitrogen stream. Detection of glycolipids was performed via TLC using a previously reported procedure⁵². MALDI-TOF analysis was used to identify glycolipids on TLC. See SI for MALDI mass spectra.

Multiple reaction monitoring profiling (MRM-Profiling) of Lipids

S. aureus ATCC 25923 (at OD₆₀₀ 0.6) was treated with DMSO, 0.25 μg/mL (1× MIC) HSGN-94, or 2 μg/mL (8× MIC) HSGN-94 for 5 hours at 37°C. The samples were normalized to OD₆₀₀ 0.9 and then centrifuged at 10,000 rpm for 10 minutes. The cells were collected and washed twice with 0.1 M sodium acetate (pH 4.7). Next, lipids were extracted following the Bligh-Dyer method⁸⁷ and lipids were isolated and dried over nitrogen stream. Experiments were carried out following a previously reported procedure⁵⁴. Briefly, data was acquired using a triple quadrupole mass spectrometer, Agilent QQQ 6410 (Santa Clara, CA), equipped with Agilent G1367A 1100 series autosampler (Santa Clara, CA). Samples were introduced into the mass spectrometer by direct flow injection ESI at a flow rate of 7 μL/min. PCA plots were constructed using MetaboAnalyst 5.0.

Macromolecular biosynthesis assay:

The inhibition of macromolecules by HSGN-94 at (0.125–4 × MIC) were assayed in triplicate via scintillation counting using the previously reported procedure⁸⁸.

MRSA murine skin infection model:

The study was approved by the Purdue Animal Care and Use Committee and carried out following the guide of the National Institutes of Health for the Care and Use of Laboratory Animals. female Balb/c mice 8-weeks old (Jackson laboratories, ME, USA), weighing on average 20 g, were used for this study. MRSA murine skin infection model was performed as described in previous reports^89–91. Brielfy, the dorsal region of each mosue mice was shaved and disinfected with ethanol and two excisional 6-mm diameter wounds were formed in each mouse using a 6 mm-diameter sterile biopsy punch (Sklar instruments, PA, USA) under isoflurane anesthesia. A subcutaneous injection of buprenorphine (0.03 mg/kg) was administered immediately before the application of biopsy punches to reduce pain. Excisional wounds were infected with 20 μL (per each wound) of 4.2×10⁹ CFU/mL of MRSA USA300. The wounds was covered with Tegaderm film (3M, MN, USA) that is fixed with Uro-Bond V silicone adhesive (Urocare, CA, USA). Two days after infection, mice were randomly divided into groups (n=5) to receive treatments. One group was injected clindamycin I.P. (25 mg/kg once daily). The formed wounds for the remaining groups received topical treatment (twice daily) with either 2% HSGN-94, petroleum jelly (vehicle), or 2% mupirocin,. After each treatment, Tegaderm was applied over the wounds. After five days of treatment, mice were humanely euthanized via CO₂ asphyxiation. The skin tissues of the wounds were harvested aseptically. For each mouse, the left wound was fixed in 10% formalin for histopathological analysis and the right wound was homogenized in sterile PBS, and plated selective MRSA plates (mannitol salt agar). The plates were incubated at 37°C before bacterial colonies were enumerated.

Expression of Proinflammatory Cytokines:

Skin homogenates obtained from the right wound of each mouse were centrifuged (10,000 rpm for 10 minutes) and the supernatant was transferred to a separate tube. For each treatment group, 100 μL aliquots from each sample (from all five mice) were pooled together. The total protein content for each sample was measured via the bicinchoninic acid assay, standardized, and expression of cytokines and growth factors was subsequently determined via the Quantibody Mouse cytokine array 4000 kit (RayBiotech Life, Norcross, GA). The concentrations of cytokines (pg/mL) in each treatment group was calculated and presented as a bar graph using GraphPad Prism 8.0 (La Jolla, CA).

Evlauation of Inflammation via Histopathology:

The left wound of infected mice in all the four groups (vehicle, HSGN-94, mupirocin and clindamycin) were evaluated histologically. Tissues were processed and sections (4-μm thick) were prepared on slides and stained with H & E. Slides were scanned using Aperio^® VERSA as a whole glass slide scanner and analyzed by a board certified veterinary pathologist.

In Silico PAINS Analysis

All synthesized compounds were assesed for PAINS using SwissADME program which demonstrated no evidence of PAINS⁹².

Abbreviations

ATCC

American type culture collection

BHIS

brain heart infusion supplemented

BOP

benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate

CLSI

clinical and laboratory standards institute

DIPEA

diisopropylethylamine

DMEM

dulbecco’s modified eagle medium

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

FBS

fetal bovine serum

MIC

minimum inhibitory concentration

MRSA

methicillin-resistant S. aureus

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)

NaOAc

sodium acetate

PAINS

pan assay interference compounds

PBS

phosphate buffered saline

RT

room temperature

TSA

tryptic soy agar

TSB

tryptic soy broth

Data Availability:

All raw LC-MS/MS data can be found in the Mass Spectrometry Interactive Virtual Environment (http://massive.ucsd.edu) with the ID: MSV000088342.