P2As are potent enhancers of penicillin G against MRSA. Certain P2As are able to reduce the MIC of penicillin G against MRSA strain ATCC BAA-1717 (Table and Supplementary Table ). The MIC of penicillin G was ~256 μg/mL, but addition of 50 μM chemical 1 enhanced the potency by at least 8-fold. A titration showed that at least 50 μM P2As was necessary to induce ≥4-fold MIC reduction for penicillin G, so these are only early hits, and will require significant optimization. P2As are able to reduce the MIC of vancomycin against VISA strain Mu50, as well. Vancomycin showed an MIC of ≤2 μg/mL against VISA in the presence of 50 μM chemical 1, when its MIC was 8 μg/mL when treated with DMSO control (Supplementary Table ). While this is a promising find, we will focus on MRSA and penicillin G in this manuscript. Recent studies have reported enhancement of oxacillin potency against MRSA strains by inhibition of Stk1 and VraS. Our findings were different, in comparison. P2As are unable to reduce the MIC of oxacillin against MRSA strain ATCC BAA-1717 (Table ). The presence of P2As did not alter the MIC of other antimicrobials, such as oxacillin, chloramphenicol and vancomycin against MRSA. Based on the above, we hypothesized that P2As function by either inhibiting penicillinases or else their expression by MRSA. Table demonstrates that penicillinase activity of live MRSA in a nitrocefin assay was indeed reduced. At the same time, a control experiment showed that 1 did not inhibit purified β-lactamase (Supplementary Table and Supplementary Fig ). The rate of nitrocefin hydrolysis was indistinguishable between two samples treated with either 1 or control. This conclusively demonstrates P2As function by suppressing penicillinase production, and not by direct neutralization. P2As bind a hypothetical, tight pocket. We have performed structure activity work (Table and to explore the space around the P2A scaffold. The pyridinyl-group at the R1 position of 1 is well tolerated, but the methylamino-substituent at the same position retains potency in 2. 3 has a methoxy-substitution, and is as potent as 1 and 2. 2 and 3 may even be marginally superior to 1, resulting in a potency increase of up to 4to 8-fold in comparison, although there was clearly some variability in our results. 4 differs from 1-2 in having no substitution at R1, but loses the ability to enhance penicillin G activity. 5, on the other hand, has a methyl-substituent at R1, and also fails to enhance penicillin G potency. Comparing 1 to 5, it seems there is an electrophilic binding partner present near the R1 position, with a propensity to bond with a nitrogen than oxygen. 4 also possesses an additional nitrogen in the core ring, making it a triazine instead of a diazine, but this does not affect activity. Chemicals 6-9 comprise the same substituents as 1 at R1 and R2, but differ at R3. None of 6-9 are able to enhance the MIC of penicillin G as well as 1; So, they help us understand the nature of the binding pocket at the R3 position. The R3 substituents are all hydrophobic, nitrogen-containing heterocyclic substituents. The 2-morpholino-2-(pyridine-2-yl)-ethyl-1-amino-substituent on 6 represents the structure closest to 1, even though effectually, it is shorter by one carbon and contains an aliphatic morpholine instead of a furan. 7, 8 and 9 have phenylamino-, piperidinyl-, and pyrrolinyl-substituents at R2, representing a shrinking substituent. Thus, comparing 6-9 with 1 suggests R3 binds to a tight pocket, almost like a narrow tunnel ending in a wide cavity. 10 is different from 6-9 because of a cyanamide substituent at R3, which is rigid due to the sp 3 carbon and nitrogen, but still lacks penicillin-enhancement activity, perhaps because it would not fit into this tunnel due to its rigid, linear nature. A few of our chemicals have concurrent substitutions at multiple positions in comparison with 1, which could explain the change in activity. 11 possesses a morpholino-group at R3 and an ethyl group at R2, even if it is indistinct from 1 otherwise. Its inability to enhance penicillin G activity could be due to both, R2 and R3 substituents. Likewise, the methyl-at R2 or the pyrrolinyl-group at R3 could contribute to reduced activity of 12. 13 represents a flexible 2-(N-methyl, N-methylsulfonyl)amino-ethyl-1amino-R3 substituent. 5 has a methyl group at R1, and a 3,3-diphenyl-propionylamide group at R3; It also fails to enhance the MIC of penicillin G, although it is uncertain whether this is due to differences at the R3 or R1 position. Most likely, a combination of both factors. 13, combined with 5, seem to indicate that simply having a flexible, hydrophobic substituent at R3 is inadequate -the 3-(furan-2-yl)-3-phenylpropyl-1-aminosubstituent present in 1-4, present at R3, seems to be required from our small sample of chemicals tested. 14 and 15 explore the R2 position with a flexible 2-hydroxy-ethyl-1amino-and a cyclic 1-pyrrolinyl-substituents that did not increase activity; both possess an unionizable amino group at R3. Neither is able to enhance the MIC of penicillin G. This could simply be due to lack of binding or failure to enhance binding -we cannot tell at this time. It should also be mentioned that, at least in theory, the R2 and R3 substituents in 14 and 15 could flip, which would explain the lack of activity of 15 (failure of a short, bulky group to bind the narrow R2 tunnel).