Head-to-Head Prenyl Tranferases: Anti-Infective Drug Targets

Abstract

We report x-ray crystallographic structures of three inhibitors bound to dehydrosqualene synthase from Staphylococcus aureus: 1 (BPH-651), 2 (WC-9) and 3 (SQ-109). Compound 2 binds to the S2 site with its –SCN group surrounded by 4 hydrogen bond donors. With 1, we report two structures: in both, the quinuclidine head group binds in the allylic (S1) site with the side-chain in S2, but in the presence of PPi and Mg²⁺, the quinuclidine’s cationic center interacts with PPi and 3 Mg²⁺, mimicking a transition state involved in diphosphate ionization. With 3, there are again two structures. In one, the geranyl side-chain binds to either S1 or S2 and the adamantane head-group binds in S1. In the second, the side-chain binds to S2, while the headgroup binds to S1. These results provide structural clues for the mechanism and inhibition of the head-to-head prenyl transferases and should aid future drug design.

INTRODUCTION

There is currently considerable interest in the structure, function and inhibition of the “head-to-head” class of prenyl tranferase enzymes that condense farnesyl diphosphate to squalene or dehydrosqualene.¹ These reactions are carried out by squalene synthase (SQS) in humans and in some protozoa such as Trypanosoma cruzi, or by dehydrosqualene synthase (CrtM) in Staphylococcus aureus (SaCrtM) (Figure 1). Inhibiting SQS in protozoa is of interest since it blocks formation of the essential membrane sterol, ergosterol, while blocking human SQS (HsSQS) lowers cholesterol levels, plus, it results in formation of anti-bacterial neutrophil extra-cellular traps (NETs)². Also of interest is the observation that inhibiting SaCrtM inhibits biofilm formation³ with S. aureus, in addition to preventing formation of the virulence factor, staphyloxanthin (Figure 1), leading to immune system based bacterial killing.⁴ There is thus interest in the development of compounds that might inhibit more than one target (e.g. combining NETs/biofilm/STX activity, or direct protozoal killing + NETs formation), and here, we present crystal structures of three SQS/CrtM inhibitors bound to CrtM, together with mechanistic insights into the SQS/CrtM mode of action. The inhibitors investigated are of interest since two are known to inhibit SQS while the third has an unknown mechanism of action but has been used in clinical trials and here, we show that it can serve as a lead for inhibiting both CrtM and SQS.

Figure 1.

Schematic illustration of the first and second half reactions catalyzed by CrtM and SQS leading to formation of dehydrosqualene and squalene, and later conversion of these products to staphyloxanthin, ergosterol and cholesterol.

RESULTS AND DISCUSSION

In the case of the quinuclidine 1^5–7 (Figure 2), a known potent squalene synthase inhibitor, we obtained two new structures of 1 bound to SaCrtM. Electron density results are shown in Figure S1A, B. In the first, Figure 3A (PDB ID code 4E9Z), 1 binds with its cationic head-group in the S1 (cationic/donor) site, while the biphenyl side-chain binds very close to S2. The cationic center is ~1.9Å from C2 in the S1 farnesyl side-chain, Figure 3A. In the second structure, Figure 3B (PDB ID code 4EA0), the side-chain occupies a site between the S1 and S2 FSPP binding sites, while the cationic center is now 2.5Å from C1, 1.7Å from C2 and 0.7Å from C3 in the (overlaid, white) S1 FSPP, Figure 3B. Both structures strongly suggest the quinuclidine is acting as an isostere for the S1 farnesyl carbo-cation. In the second structure (PDB ID code 4EA0) there is also a PPi group as well as three Mg²⁺, with the (inorganic) PPi being close to the position occupied by the diphosphate group in the S1 FSPP structure,⁴ plus, two of the three Mg²⁺ (Mg_A²⁺, Mg_B²⁺) seen in the FSPP structure are observed, Figure 3B. These results are consistent with the proposal that the initial FPP ionization step occurs in the S1 site,⁸ and are reminiscent of the observation that cationic bisphosphonate inhibitors of other prenyl synthases, such as FPP synthase (FPPS), have their cationic, anionic and Mg²⁺ located in very similar positions, as shown in the 4/FPPS⁹ superimposition shown in Figure 3C. Notably, this S1/cationic site structure is quite distinct from that reported previously for 1 (via soaking, as opposed to co-crystallization), in which the quinuclidine carbo-cation feature was located in essentially the same position as the cyclopropane ring in PSPP, Figure 3D, where it mimics a second transition state. So, the quinuclidine (1) can block either the S1 or S2 head-group sites, opening up new possibilities for inhibitor design.

Figure 2.

Chemical structures of small molecule inhibitors.

Figure 3.

X-ray crystallographic structures of 1 bound to CrtM. (A) CrtM-1 (no Mg²⁺, PPi; PDB ID code 4E9Z). Cationic (N⁺) center (blue) is close to S1 FPP C3. FSPP superimposed in white. (B) CrtM-1 + Mg²⁺ + PPi (PDB ID code 4EA0). Cationic center again close to putative S1 allylic carbocation; N⁺ also close to PPi group. (C) As (B) but superimposed on zoledronate/Mg²⁺ ligand from FPPS structure (PDB ID code 2F8C). (D) 1 occupying S2 carbocation center (PDB ID code 3ACW), superimposed on PSPP structure (PDB ID code 3NPR). (E) Structure from B superimposed on 6/Mg²⁺/PPi from epi-isozizaene synthase (PDB ID 3KB9). (F) Top view of B showing Y129-H₂O-Mg_D²⁺ and PPi-Mg²⁺ interactions. The ligand is s-thiolo farnesyl diphosphate.

The 1-PPi-[Mg²⁺]₃ structure (Figure 3B) is also of considerable interest since it bears a strong resemblance - in terms of placement of the cationic center, PPi and [Mg²⁺]₃ groups – to the recently reported structure¹⁰ of the terpene cyclase, epi-isozizaene (5) synthase, containing a bound inhibitor, 6 (benzyltriethylammonium, Figure 2). As can be seen in the superposition shown in Figure 3E, both structures lack the [Mg_C²⁺] seen in the CrtM/FSPP structure⁴ and contain instead, a new Mg²⁺, Mg_D²⁺, Figures 3E, F. The rmsd of the N⁺, PPi and 3 Mg²⁺ in the CrtM and epi-isozizaene structures is only 0.35Å, supporting the idea that diphosphate ionization of FPP in the head-to-head prenyl transferases, as well as in the terpene cyclase, is dominated by the same driving force, a [Mg²⁺]₃-PPi interaction. The results obtained with the 1-PPi-[Mg²⁺]₃ structure are also of interest since they help clarify the role of Y129 in CrtM (Y171 in HsSQS), which is among the most essential residues needed for catalytic activity (based on mutagenesis^{8, 11} and a SCORECONS analysis¹²). In earlier work, it was thought that this residue (in HsSQS) might be involved in stabilizing the farnesyl cation via a cation-π interaction, however, this residue is ~8.5Å from the proposed cationic center. In the 1-PPi-[Mg²⁺] structure, we now see that the Tyr-OH is hydrogen bonded to a water molecule that coordinates to one of the Mg²⁺ seen in the x-ray structure, Mg_D²⁺, Figure 3F (in blue). This suggests that Y129 may help stabilize and/or facilitate removal of the diphosphate group, rather than directly stabilizing the S1 carbo-cation.

We show in Figure 4A, B the single crystal x-ray crystallographic structure of the thiocyanate 2¹³ bound to CrtM. 2 is a potent SQS inhibitor of interest in treating Chagas disease.^{14, 15} Electron density results are shown in Figure S1C and full crystallographic data acquisition and structure refinement results are in Table 1. The molecule inhibits CrtM with a K_i= 1.5 μM (Figure S2A) and binds with its diphenyl ether side-chain occupying the S2 site normally occupied by the acceptor FPP, or one of the PSPP side-chains, as seen in the 2 (cyan) – PSPP (yellow) superposition in Figure 4B. This side-chain binding site can also be occupied by several other inhibitors, including the phosphonosulfonates,⁴ and is completely hydrophobic. The question then arises as to the nature of the interactions undergone by the thiocyanate group. Unlike the quinuclidine inhibitors, the thiocyanate group cannot be charged, however, alkyl thiocyanates can act as proton acceptors, due to the following resonance scheme:

$$\text{R}-\text{S}-\text{C}\equiv \text{N}\leftrightarrow {\text{R}-\text{S}}^{+}=\text{C}={\text{N}}^{-}$$

and there is a ΔH = −6.5 kJ mol⁻¹ interaction between phenol and CH₃SCN.¹⁶ In the 2/CrtM crystal structure (PDB ID code 4E9U), there are four polar residues close to the thiocyanate nitrogen (Y41, Q165, N168 and Y248) with, on average, an SCN-protein distance of ~3.2Å, Figure 4B. Since all of these amino-acid side-chains are polar, it seems likely that they will contribute to ligand binding via electrostatic (hydrogen bonding) interactions, in much the same way that e.g. phenol interacts with the thiocyanate group in liquid MeSCN.¹⁶

Figure 4.

X-ray crystallographic structure of 2 bound to CrtM (PDB ID code 4E9U). (A) 2 binds to a buried, hydrophobic S2 site. (B) 2 (in cyan) superimposed on the PSPP reaction intermediate (yellow) bound to CrtM.

Table 1.

Data collection and refinement statistics for CrtM with 1, 2, and 3

Crystal (PDB ID)	2/CrtM (soaking) (4E9U)	1/CrtM (co-crystal) (4E9Z)	1/CrtM -PPi- Mg2+ (co-crystal) (4EA0)	3/CrtM (co-crystal) (4EA1)	3/CrtM (soaking) (4EA2)
Data collection
Radiation source	APS 21-ID-F	APS 21-ID-F	APS 21-ID-F	APS 21-ID-F	APS 21-ID-F
Wavelength (Å)	0.97857	0.97857	0.97857	0.97857	0.97857
Space group	P3221	P32 2 1	P31 2 1	P32 2 1	P32 2 1
a(Å)	80.4	80.6	80.3	80.6	80.1
b(Å)	80.4	80.6	80.3	80.6	80.1
c (Å)	90.8	91.8	180.8	91.6	90.9
Resolution (Å)a	50.0-2.10 (2.14-2.10)	50.0-2.06 (2.12-2.06)	50.0-2.12 (2.17-2.12)	50.0-2.46 (2.50-2.46)	50.0-2.02 (2.09-2.05)
No. of reflections	20218 (966)	21572 (1041)	37084 (1169)	12795 (583)	21470 (858)
Completeness (%)	99.9 (98.9)	99.9 (100)	94.5 (61.1)	99.2 (91.0)	98.5 (80.9)
Redundancy	10.1 (4.90)	12.7 (12.7)	8.8 (6.1)	9.5 (5.4)	10.1 (5.0)
Rmerge (%)	10.1 (65.2)	7.30 (45.0)	7.60 (59.7)	8.80 (44.1)	6.5 (68.9)
I/s(I)	21.3 (1.56)	44.0 (5.04)	27.7 (1.52)	2.69 (1.74)	41.8 (1.5)
Refinement
Resolution (Å) a	36.8-2.1 (2.21-2.10)	30.0-2.08 (2.12-2.06)	50.0-2.12 (2.17-2.12)	30.0-2.46 (2.17-2.12)	34.7-2.05 (2.10-2.05)
No. of reflections	19540 (2320)	20373 (1228)	35058 (1687)	12091 (819)	20270 (1246)
Rwork (%)	18.9 (21.8)	19.1 (20.8)	22.2 (31.5)	23.2 (34.8)	20.6 (27.2)
Rfree (%)	23.6 (26.5)	24.1 (24.7)	27.8 (36.3)	31.4 (34.8)	26.0 (31.7)
Geometry deviations
Bond lengths (Å)	0.007	0.015	0.005	0.006	0.014
Bond angles (°)	0.962	1.331	0.692	0.912	1.234
Mean B-values (Å2) / Number of non-H atoms
All refined atoms	38.5 / 2568	34.0 / 2602	46.2 / 5046	42.3 / 2456	48.76 / 2467
Compound atoms	49.7 / 19	58.1 / 21	38.4 / 42	34.9 / 48	71.8 / 24
Mg ions	-	-	28.5 / 6	-	75.6 / 1
Water molecules	38.5 / 156	40.8 / 184	41.6 / 186	40.6 / 47	52.4 / 63
Ramachandran plot (%)
Most favored	98.6	98.2	96.6	96.4	96.8
Additionally allowed	1.06	1.07	2.84	1.8	2.2
Generously allowed	0.35	0.71	0.53	1.8	1.0

^aValues in the parentheses are for the highest resolution shells.

The question then arises as to whether 2 binds to Trypanosoma cruzi SQS (TcSQS) in the same manner as it does to CrtM. To date, there are no structures of TcSQS. However, there are 11 residues in CrtM (F22, Y41, A134, V137, G138, L141, A157, G161, L164, Q165 and N168) that are close (< 4Å) to 2 in the CrtM structure, and these residues are totally conserved in both HsSQS as well as TcSQS. This strongly suggests that the ligand will bind into the same S2 pocket in TcSQS, with the same polar interactions with the thiocyanate group as in CrtM.

Finally, we determined two structures of 3 bound to CrtM. 3 is a novel, di-alkylated ethylendiamine with an adamantyl “head-group” and a geranyl “side-chain” that potently inhibits the growth of Candida albicans, Aspergillus fumigatus, Mycobacterium tuberculosis and Helicobacter pylori and has progressed through Phase Ia clinical trials.¹⁷ The actual enzyme targets involved have not been reported. Nevertheless, based on the general similarities (large hydrophobic group--cation center--small hydrophobic group) between the quinuclidine (1) and 3 structures it seemed possible that 3 might inhibit CrtM and SQS. This is the case, with K_i values of 0.36 μM (CrtM), 0.74 μM (hSQS), and 1.2 μM (TcSQS), Figure S2. We show in Figure 5A two crystallographic structures of 3 bound to CrtM, one obtained by co-crystallization (PDB ID code 4EA1), the other by soaking (PDB ID code 4EA2). Electron density results are shown in Figure S1D, E. In the co-crystal structure, we found two alternative conformations, with the geranyl side-chain binding to either the S1 or S2 sites, the ethylenediamine linker interacting with either D48 or Q165, and the adamantane head-group being solvent exposed (Figures S3, 4). In the structure obtained by soaking, 3 adopts a “bent” conformation with the adamantane head-group binding to the S1 site, close to the position occupied by the quinuclidine head-group in 1, and the geranyl side-chain is located in the S2 site, Figure 5B and S5. In this structure, the hydrophobic head-group occupies the same pocket as occupied by the “bend” in presqualene diphosphate (or FSPP), as can be seen in the superimposition shown in Figure 5B (PSPP in magenta). Since 3 has been successfully tested in humans, the observation that it inhibits both human and T. cruzi SQS as well as S. aureus CrtM clearly makes it an interesting potential lead for further development, as an SQS/CrtM inhibitor.

Figure 5.

X-ray crystallographic structure of 3 bound to CrtM. (A) Structure obtained by co-crystallization (PDB ID code 4EA1) showing geranyl side-chain can occupy both S1 and S2 sites (cyan, S1; magenta, S2), superimposed on the FSPP structures (green, yellow; PDB ID code 2ZCP). (B) Structure obtained by soaking (PDB ID code 4EA2). The inhibitor 3 (cyan) adopts a bent conformation with the adamantane head-group occupying the same site as the “bend” in the PSPP (magenta) intermediate, in S1 (PDB ID code 3NPR).

CONCLUSIONS

The results presented here are of interest for several reasons. First, we have obtained the structure of the potent anti-parasitic thiocyanate drug lead 2, that inhibits squalene synthase, bound here to a dehydrosqualene synthase (Figure 4). The biphenyl ether side-chain binds to the S1 site and the thiocyanate group is involved in an extensive series of electrostatic interactions with protein residues. There are 11 residues in close contact with the ligand and these residues are all conserved in S. aureus CrtM, human SQS as well as T. cruzi SQS, supporting binding of this inhibitor to the S2 pocket the SQS enzymes as well. Second, we report two structures of the quinuclidine (1), bound to dehydrosqualene synthase. One structure (1+PPi/Mg) is of particular interest since it mimics a transition state in which the cationic center formed on FPP (or PSPP) ionization is in S1, while the exiting PPi binds to a new [Mg²⁺]₃ cluster, with one Mg²⁺ close to the highly conserved Y129, which we propose is positioned to facilitate PPi removal. Third, we find that the anti-infective drug 3 inhibits both CrtM as well as SQS (Figure S2). We obtained two CrtM structures. In one, the geranyl side-chain binds to either the S1 or S2 sites, the ethylenediamine interacts with essential protein residues, and the adamantane group is solvent exposed (Figure 5A). In the second (Figure 5B), the geranyl side-chain occupies the S2 site, while the adamantane group is now buried, and occupies the same position as the “bend” in the S1 PSPP sidechain.⁸

Overall, these results are of significance since they provide new structural information on the mechanism of action and inhibition of the head-to-head prenyl transferases that are of interest as novel anti-infective drug targets. Such inhibitors may target not only direct pathogen killing,^{6, 13, 18} but also innate immune system-based killing, via virulence factor inhibition,⁴ NET formation² and bio-film inhibition.³

EXPERIMENTAL SECTION

Materials

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Alchem Laboratories Corp. and were used as provided. BL-21 (DE3) competent cells were purchased from Stratagene (La Jolla, CA). Syntheses of 1–3 (Figure 2) were as reported previously.^{15, 19, 20} The CrtM plasmid was provided by Andrew H.-J. Wang’s group. The human SQS (HsSQS) and Trypanosoma cruzi SQS (TcSQS) plasmids were provided by Dolores Gonzalez-Pacanowska.

Protein Expression, Purification and Crystallization

Protein expression, purification and crystallization were as described previously.^{4, 6, 8}

Inhibition Assays

CrtM, TcSQS and hSQS inhibition assays were as described previously.^{4, 8}

Crystallographic Aspect

Structures were obtained as described in detail previously.^{4, 8} Structure Figures were made by using PyMOL²¹ and ligand-protein interaction Figures were made using MOE.²²

The abbreviations used are

CrtM

dehydrosqualene synthase

SQS

squalene synthase

FPP

farnesyl diphosphate

FPPS

farnesyl diphosphate synthase

FSPP

S-thiolo-farnesyl diphosphate

PSPP

presqualene diphosphate

PPi

diphosphate

BTAC

benzyltrimethylammonium

NET

neutrophil extra-cellular trap