HIV-1 Integrase Inhibitor-Inspired Antibacterials Targeting Isoprenoid Biosynthesis

Abstract

We report the discovery of antibacterial leads, keto- and diketo-acids, targeting two prenyl transferases: undecaprenyl diphosphate synthase (UPPS) and dehydrosqualene synthase (CrtM). The leads were suggested by the observation that keto- and diketo-acids bind to the active site Mg²⁺/Asp domain in HIV-1 integrase, and similar domains are present in prenyl transferases. We report the x-ray crystallographic structures of one diketo-acid and one keto-acid bound to CrtM, which supports the Mg²⁺ binding hypothesis, together with the x-ray structure of one diketo-acid bound to UPPS. In all cases, the inhibitors bind to a farnesyl diphosphate substrate-binding site. Compound 45 had cell growth inhibition MIC₉₀ values of ~250–500 ng/mL against S. aureus, 500 ng/mL against Bacillus anthracis, 4 μg/mL against Listeria monocytogenes and Enterococcus faecium, and 1 μg/mL against Streptococcus pyogenes M1, but very little activity against E. coli (DH5α, K12) or human cell lines.

There is currently an urgent need for new types of anti-bacterials exhibiting novel modes of action, due to the rapid rise in drug resistance,¹ and isoprenoid biosynthesis² is one attractive target. For example, cell wall biosynthesis can be inhibited by targeting farnesyl diphosphate synthase (FPPS) or undecaprenyl diphosphate synthase (UPPS), involved in lipid I biosynthesis (Figure 1). In addition, in S. aureus, formation of the virulence factor staphyloxanthin³ can be blocked by inhibiting dehydrosqualene synthase (CrtM), resulting in a lowering of the anti-oxidant shield to host derived ROS⁴ (Figure 1). The bisphosphonate class of drugs such as zoledronate (1, Chart 1) are potent, low nM inhibitors of FPPS, but 1 has little antibacterial activity (due presumably to lack of cell penetration), although more lipophilic bisphosphonates such as 2 (BPH-210, Chart 1) have modest activity (IC₅₀ ~30 μM) against E. coli.⁵ More lipophilic bisphosphonates also potently target UPPS,⁶ as well as CrtM⁴, but again they have essentially no activity in bacteria. Replacing one phosphonate group by a sulfonate to form a phosphonosulfonate results, however, in potent CrtM inhibitors (e.g. 3, BPH-652, Chart 1, IC₅₀ ~ 7.9 μM, K_i ~ 80 nM) that also blocks carotenoid pigment formation in cells (IC₅₀ ~110 nM).⁷ In addition, there has recently been interest in developing phosphorus-free prenyl transferase inhibitors, which might have even more drug-like properties. For example, Jahnke et al. reported a series of FPPS inhibitors, dicarboxylic acids, that bound to a novel, allosteric site.⁸ In addition, other species such as tetramic acid UPPS inhibitors have been described (e.g. 4, Chart 1),⁹ but to date their x-ray structures have not been reported, although an allosteric model has been proposed.¹⁰

Figure 1.

Biosynthetic reactions catalyzed by CrtM and UPPS, with the end products of the pathways shown.

Chart 1.

Chemical structures of selected compounds and the inhibition of CrtM, E. coli UPPS and S. aureus UPPS by 7, 8, 9, 41, 42, 44, 45.

A key component of the active site of most prenyl transferases is a Mg²⁺/Asp motif that interacts with a substrate's diphosphate group. We reasoned that HIV-1 integrase (IN) inhibitors¹¹ might provide clues for new prenyl transferase inhibitors, since IN contains a similar Asp/Mg²⁺ motif¹² and IN inhibitors such as 5 (L-708,906, Chart 1)¹³ and 6 (elvitegravir, Chart 1)¹⁴, diketo-acids and keto-acids, respectively, are thought to bind at or near the Mg²⁺/Asp motif in the IN active site.¹⁵ In addition, many other IN inhibitors like raltegravir, dolutegravir, MK2048 etc (structures not shown) have been found to bind Mg²⁺.^{15b, 16}

We thus made a small screening library (38 compounds) of IN inhibitor-inspired molecules and their structures and inhibition of S. aureus CrtM, E. coli UPPS and S. aureus UPPS are shown in Figure S1. Most compounds were amide-diketo acids (7–40, class I, Figure S1) and were conveniently prepared from the synthon (Z)-2,2-dimethyl-5-carboxymethylene-1,3-dioxolan-4-one¹⁷ by amine coupling. Among these compounds, 7 (Chart I) inhibited CrtM with IC₅₀ ~24 μM, K_i ~ 250 nM (for comparison, K_i of 3 ~ 70 nM⁷), and blocked staphyloxanthin pigment formation (IC₅₀ = 4 μM). Inhibitors of Class II were keto-acids, dihydropyridone-3-carboxylates, and were based on 6 (Elvitegravir) and dihydroquinoline-3-carboxylic acid IN inhibitors¹⁸, which again are thought to bind via their carboxyl and carbonyl oxygens to Mg²⁺/Asp.¹⁹ We made two analogs, 41, 42 (Chart I), with alkoxy-aryl tails to mimic the substrate FPP. The longer-chain species 42 had no activity, but the shorter chain species 41 had a CrtM IC₅₀= 45 μM, K_i = 450 nM and a loss of pigmentation IC₅₀ of 33 μM.

To see how these inhibitors bound to CrtM, we carried out co-crystallization and soaking experiments with 7 (Class I) and 41 (Class II) and obtained crystals (by soaking) that diffracted to 2.3 Å and 1.9Å, respectively. Full x-ray crystallographic data and structure refinement details are given in Supporting Information, Table S1. Electron density results for 7 are shown in Figure 2a and indicate the presence of 7 in addition to one molecule of farnesyl monophosphate (FMP) that co-purified with the protein. The identity of FMP was further confirmed by LC-MS (Supporting Information, Figure S2) and the electron density results (Figure 2a). The diphenyl ether fragment in 7 (cyan) binds into the CrtM S1 site²⁰, and is shown in Figure 2b superimposed on one of the S-thiolo-farnesyl diphosphate (FSPP) inhibitors (in yellow, green) whose structures were reported previously.⁴ This binding mode is similar to that seen with the phosphonosulfonate 3 (Figure 2c), with the diketo-acid head-group interacting with two of the three Mg²⁺ (Mg²⁺_B,C) seen in the CrtM-FSPP structure (Figure 2d). The farnesyl side-chain in FMP bound to the S2 site and had a 0.8 Å rmsd from the S2 FSPP reported previously.⁴ With 41, the ligand electron density is again well defined (Figure 3a), and the crystallographic results show the side-chain binds in S2, similar to the farnesyl side-chain in the FSPP structures (Figure 3b), as well as the phosphonoacetamide analog of 7 (43, BPH-830²¹, Figure 3c). There are 3 Mg²⁺ in the x-ray structure. However, these are not the Mg²⁺_ABC seen in most prenyl transferases²² but rather, Mg²⁺_BCD. That is, there is a new Mg²⁺ binding site, Mg²⁺_D. The dihydropyridone side-chain interacts with Mg²⁺_CD but surprisingly, via the two ring oxygens, not the carboxylate (Figure 3d), which interacts with two water molecules (Supporting Information, Figure S3). These inhibition and structural results for 7 and 41 clearly support the Mg²⁺ binding hypothesis, at least for CrtM.

Figure 2.

CrtM crystallographic structures. a) 7 (cyan) plus FMP (yellow) electron density, bound to CrtM. b) 7 (cyan) bound to CrtM, superimposed on two FSPP molecules (yellow, green; PDB ID code 2ZCP). Also shown is the farnesyl monophosphate (magenta) that co-crystallized. The Mg²⁺ are from the FSPP structure. c) Comparison between 7 (cyan) and 3 (magenta, PDB ID code 2ZCQ) bound to CrtM. Both diphenyl ether side-chains bind in S1. d) Interactions between 7 (cyan), FMP (magenta) and Mg²⁺ in CrtM.

Figure 3.

CrtM crystallographic structures. a) Electron density of 41 bound to CrtM. b) Structure of 41 (cyan) bound to CrtM shown superimposed on two FSPP molecules (yellow, green). c) Comparison between 41 (cyan) and the phosphonoacetamide analog of 7(compound 43, BPH-830)²¹ (magenta) bound to CrtM (PDB ID code 2ZY1). d) Interactions between 41 (cyan) and Mg²⁺ in CrtM.

CrtM is a so-called head-to-head prenyl transferase so we next sought to see if any of the molecules synthesized might also inhibit the head-to-tail prenyl transferase FPPS, or the cis-prenyl transferase, UPPS. There was no activity against FPPS (probably due to the lack of a positively charged feature that mimics the carbocation involved in FPP biosynthesis) but most of the amide-diketo acids (Class I) were potent UPPS inhibitors with the most active one (8) having an IC₅₀ ~ 240 nM and K_i ~ 120 nM, comparable to the most active bisphosphonate UPPS inhibitor BPH-629 (IC₅₀ ~ 300 nM for E. coli UPPS)⁶. There are four different ligand-binding sites in UPPS (designated 1–4 in Ref 6) found with bisphosphonate inhibitors. This is not unexpected since the UPPS product, undecaprenyl diphosphate (UPP) contains 55-carbon atoms and is thus much larger than the (C₁₅) FPP substrate. In principle, then, novel inhibitors might occupy multiple binding sites.

Co-crystallization of E. coli UPPS with 9 (IC₅₀ = 560 nM) produced well-formed crystals with E. coli UPPS, and the electron density was well resolved (Figure 4a). As can be seen in Figure 4b, 9 binds to site 1⁶, the FPP binding site and, as can be seen in Figure 4c, 9 (in cyan) closely maps the FPP backbone structure (in yellow) with the diketo-acid fragment being located close to two of the three most essential residues in UPPS, D26 and N28 (Figure 4d). We found no evidence for the presence of Mg²⁺, but this observation is not entirely unexpected since even with the 5 E. coli UPPS x-ray structures with strong Mg²⁺ chelators – bisphosphonates (PDB ID codes 2E98, 2E99, 2E9A, 2E9C, 2E9D),⁶ Mg²⁺ was not observed.

Figure 4.

UPPS crystallographic structures. a) Electron density of 9 bound to UPPS. b) Structure of 9 (cyan) bound to UPPS, superimposed on FSPP/Mg²⁺ (from PDB ID code 1X06) and 4 bisphosphonate inhibitors (PDB ID code 2E98). c) Superposition of 9 (cyan) on FSPP (yellow) in site-1 in UPPS. The Mg²⁺ is from the FSPP structure. d) The diketo-acid head-group of 9 binds into the active site of UPPS and interacts with D26 and N28.

The amide-diketo acids were not growth suppressive towards S. aureus or E. coli, perhaps due to the instability of the amide bond inside the cells, or a lack of cell permeability. However, 44 and 45 (aryldiketo acids, Class III), had good activity against S. aureus UPPS (44, IC₅₀ = 0.73 μM, K_i = 230 nM; 45, IC₅₀ = 2.0 μM, K_i = 670 nM) and both were active against the USA300 (MRSA) strain of S. aureus with MIC₉₀ values of 500 ng/mL (44) and 250–500 ng/mL (45). There was no appreciable activity against the Gram-negative E. coli, however, there was promising activity against other Gram-positives: ~ 500 ng/mL against Bacillus anthracis str. Sterne, ~ 4 μg/mL against Listeria monocytogenes and Enterococcus faecium U503, and ~ 1 μg/mL for Streptococcus pyogenes M1. While the precise mechanism of action of these compounds in each cell remains to be determined, UPPS inhibition is a likely candidate. In addition, we found low toxicity against a human cell line (MCF-7; IC₅₀ ~30 μM), consistent with poor FPPS inhibition.

These results are important for several reasons. First, we tested the hypothesis that keto- and diketo-acids might inhibit prenyl transferase enzymes, based on the presence of Mg²⁺/Asp motifs in their active sites – an “integrase inhibitor-inspired” approach. The best CrtM inhibitors had K_i ~ 250 nM and were active in blocking staphyloxanthin biosynthesis in S. aureus, and we solved two structures of lead compounds bound to CrtM. In both, the inhibitor head-groups bound to Mg²⁺, while the side-chains bound to one or the other of the two FPP side-chain binding sites. Second, we tested this small library for FPPS and UPPS inhibition. There was no FPPS inhibition, but the most potent UPPS inhibitor had an IC₅₀ = 240 nM, and we determined the structure of one such lead bound to E. coli UPPS – the first UPPS x-ray structure reported for a non-bisphosphonate inhibitor. We also found low toxicity and promising activity against a subset of Gram-positive bacteria with MIC₉₀ values as low as 250–500 ng/mL against USA300 S. aureus and 500 ng/mL against Bacillus anthracis str. Sterne, and low activity against E. coli and a human cell line. Overall, these results indicate that integrase-inspired inhibitors may be engineered into drug leads that target isoprenoid biosynthesis.

ABBREVIATIONS

CrtM

dehydrosqualene synthase

UPPS

undecaprenyl diphosphate synthase

FPPS

farnesyl diphosphate synthase

FPP

farnesyl diphosphate

(FMP)

farnesyl monophosphate

FSPP

S-thiolo-farnesyl diphosphate

IN

HIV-integrase