Novel Architectures for Multicationic Quaternary Ammonium Compounds (multiQACs) Saleh Al-Khalifa, Megan Jennings, William M. Wuest, and Kevin P.C. Minbiole Department of Chemistry, Villanova University, Villanova, Pennsylvania

Abstract

The emergence of bacterial resistance toward commonly used antimicrobial agents is a pressing concern for human health. The lack of investigation into this antiseptics trend may lead to serious consequences in the future. Therefore, it is important as scientists to develop a deeper understanding of the mechanisms by which resistance proliferates, as well as potential means for mitigation. While longstanding antiseptic quaternary ammonium compounds (QACs) such as benzalkonium chloride provide a single cationic nitrogen to confer amphiphilicity, we have observed that multicationic structures are more effective in their ability to kill bacteria and eradicate biofilms. It has been found that these “multiQACs” show little or no susceptibility to bacterial resistance, which is capable of reducing the efficacy of many quaternary ammonium compounds. We have synthesized 54 novel QACs based on a scaffold hopping approach. The preparation of these structures allowed for the correlation of scaffold structure with antimicrobial activity. We also confirmed that resistance, as evidenced by an increased minimum inhibitory concentration (MIC) for methicillin-resistant Staphylococcus aureus (MRSA) compared to methicillin-susceptible Staphylococcus aureus (MSSA), can reduce efficacy up to 64-fold versus monocationic QACs.

Introduction

Amphiphiles, compounds bearing polar and non-polar regions, are of great importance due to their ability to act as effective antimicrobial agents.1 The detergent-like mechanism of action2 that quaternary ammonium compounds exhibit initially stems from the cationic charge that they possess, which adheres to the bacterial cell surface; the hydrophobic regions of the QAC disrupt the bacterial cell membrane, thus resulting in cell lysis.2,3 With such a fundamental target, a belief arose that QACs would be immune to bacterial resistance. However, recent developments have proven this claim to be inaccurate – the resistance to QACs has been increasing at an astounding rate; this is due to a number of bacterial resistance mechanisms.4 In order to overcome QAC-resistant bacteria, new generations of QACs must be developed that target a more selective mechanism of action for microbial cells rather than a more global mechanism of action for eukaryotic cells.2

Biological Evaluation of Select multiQAC Compounds

Acknowledgments This work was supported by Villanova University & Temple University, as well as a grant from the University City Science Center (QED program).

References

Conclusions & Future Work These series of QACs with varied cationic character were synthesized in order to investigate

what structural features would optimally inhibit growth of bacterial strains. It was found that permanent charge (up to 4+) as well as alkyl length effect efficacy and susceptibility to resistance.7 These scaffolds are unlikely to trigger QAC resistance, as evidenced by the minimal resistance observed (comparing MSSA to MRSA MIC values). With multiple amphiphiles displaying sub-micromolar MICs, a novel set of potent antiseptics has indeed been prepared. Future work in our laboratory will use these compounds in chemical genetic approaches to better understand how multiQACs are able to evade resistance.

Compound Derivatization

1. Mitchell, M.A., Iannetta, A.A, Jennings, M.C., Fletcher, M.H., Wuest, W.M., and Minbiole, K.P.C. (2015) Scaffold-hopping of Multicationic Amphiphiles Yields Three New Classes of Antimicrobials. ChemBioChem, 2299-2303. 2. Jennings, M.C.; Minbiole, K.P.C.; Wuest, W.M. (2015) Quaternary Ammonium Compounds: An Antimicrobial Mainstay and Platform for Innovation to address Bacterial Resistance. ACS Inf. Dis. 1, 288-303. 3. Paniak, T.J., Jennings, M.C., Shanahan, P.C., Joyce, M.D., Santiago, C.N., Wuest, W.M., and Minbiole, K.P.C. (2014) The Antimicrobial Activity of Mono-, Bis-, Tris-, and Tetracationic Amphiphiles Derived From Simple Polyamine Platforms. Bioorg. Med. Chem. Lett. 5824-5828. 4. Centers for Disease Control and Prevention. Antibiotic 1149 Resistance Threats in the United States; 2013, pp 1−113. 5. Schumacher, M.A., Miller, M.C., Grkovic, S., Brown, M.H., Skurray, R.A., and Brennan, R.G. (2001) Structural mechanisms of QacR induction and multidrug recognition. Science 294, 2158-2163. 6. Jennings, M.C.; Buttaro, B.A.; Minbiole, K.P.C.; Wuest, W.M. (2015) Bioorganic Investigation of Multicationic Antimicrobials to Combat QAC-Resistant Staphylococcus aureus. ACS Infectious Diseases 1, 304-309. 7. Forman, M.E., Fletcher, M.H., Jennings, M.C., Duggan, S.M., Minbiole, K.P.C., Wuest, W.M. (2016) Structure-Resistance Relationships: Interrogating Antiseptic Resistance in Bacteria with Multicationic Quaternary Ammonium Dyes. ChemBioChem. 11, 1401-1405.

QAC Mechanism of Action

The permeation of the QAC side chains into the intramembrane region followsthe electrostatic interactions that occur between the positively charged QAC head and negative charged cell membrane of the bacteria. Once in the membrane, a leakage of cytoplasmic material occurs resulting in cellular lysis (Figure 1).2

Table 1. Minimum Inhibitory Concentrations (MIC) in µM of multiQAC analogs.

Figure 4. Synthetic Schemes for P-Series1

Figure 1. Mechanism of Action of QACs2

Compounds of Interest

N NN

N

N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethylpropane-1,3-diamine

Tris(2-dimethylaminoethyl)amine

NNN

N

Bacterial Resistance Mechanism of Action

Figure 2. Resistance Mechanism to QACs2

Due to the exposure and overuse of traditional antimicrobial agents, bacteria have proven to activate resistance mechanisms. QACs are able to passively diffuse through the cell membrane at sub-MIC-concentrations. Once in the cell membrane, the QACs are then able to bind QacR, a negative transcriptional regulator of qacA, a membrane affiliated efflux pump.2 QacR induces QacA production which uses proton motive force to combat mono- and bis-QACs in the cell membrane thus rendering them ineffective as antibacterial agents (Figure 2).2,5 With the development of new multiQACs, bacteria must find a new mechanism different than that of qacA/R in order to develop resistance.6

LinkforC-series

LinkforP-series AddamineforT-series

Figure 5. Synthetic Schemes for C-Series1

Figure 6. Synthetic Schemes for T-Series1

Figure 7. Synthetic Schemes for superT-Series7

P-0,0,0 P-n,n,n

C-0,0,0 C-n,0,0 C-n,1,1

T-0,0,0 T-n,n,n T-n,n,n,1

sT-0,0,0 sT-n,n,n

sT-n,n,n,Bz

sT-n,n,n,1

Figure 3. Scaffold-hopping Approach: Key Starting Materials

ExtendlinkersforsuperT-series

superT-series

T-series

P-series

C-series

MIC(μM)Compound S.Aureus

(MSSA)USA300-0114

(MRSA) E.coli P.aeruginosa Lysis20

STD BenzalkoniumChloride 8 32 32 63 63

PSerie

s

P-10,0,10 1 32 16 63 125P-11,0,11 0.5 2 1 8 16P-12,0,12 0.5 0.5 1 2 8P-13,0,13 1 1 1 4 8P-14,0,14 0.5 0.5 1 8 4

CSerie

s

C-11,0,0 8 125 63 500 ≥500C-12,0,0 2 63 32 250 250C-13,0,0 1 16 16 125 32C-14,0,0 0.5 8 8 63 32C-16,0,0 ≤0.25 8 4 16 16C-11,1,1 63 32 250 125 250C-12,1,1 16 16 125 250 250C-13,1,1 4 8 16 125 125C-14,1,1 2 4 8 32 63C-16,1,1 1 1 1 16 16

TSerie

s

T-10,10,10 1 1 1 2 8T-11,11,11 0.5 1 1 2 8T-12,12,12 1 2 2 8 8T-14,14,14 4 32 16 63 8T-16,16,16 NT NT NT NT NT

Supe

rT

sT-11,11,11,0 0.5 0.5 0.5 1 4sT-12,12,12,0 0.5 0.5 1 4 4sT-13,13,13,0 1 0.5 1 8 4sT-14,14,14,0 1 0.5 4 16 4sT-16,16,16,0 8 4 16 32 4sT-10,10,10,1 0.5 0.5 0.5 2 8sT-11,11,11,1 0.5 0.5 1 1 8sT-11,11,11,1 0.5 0.5 0.25 1 4sT-12,12,12,1 1 0.5 1 4 16sT-13,13,13,1 1 1 2 8 4sT-13,13,13,1 2 0.5 1 4 4sT-14,14,14,1 4 2 4 32 4sT-8,8,8,3A 1 1 4 125 125

sT-10,10,10,3A 0.5 0.25 0.5 1 8sT-11,11,11,3A 0.5 0.5 0.5 1 4sT-12,12,12,3A 1 1 0.5 2 4sT-13,13,13,3A 2 1 1 4 2sT-14,14,14,3A 2 2 4 32 2sT-16,16,16,3A 4 4 16 63 4sT-18,18,18,3A 2 2 32 125 4sT-11,11,11,Bn 0.5 0.5 0.5 1 4sT-12,12,12,Bn 1 0.5 0.5 2 4