Targeting isoprenoid biosynthesis for drug discovery: bench to bedside

Abstract

The isoprenoid biosynthesis pathways produce the largest class of small molecules in Nature: isoprenoids (also called terpenoids). Not surprisingly then, isoprenoid biosynthesis is a target for drug discovery, and many drugs--such as Lipitor (used to lower cholesterol), Fosamax (used to treat osteoporosis), and many anti-infectives--target isoprenoid biosynthesis. However, drug resistance in malaria, tuberculosis, and staph infections is rising, cheap and effective drugs for the neglected tropical diseases are lacking, and progress in the development of anticancer drugs is relatively slow. Isoprenoid biosynthesis is thus an attractive target, and in this Account, I describe developments in four areas, using in each case knowledge derived from one area of chemistry to guide the development of inhibitors (or drug leads) in another, seemingly unrelated, area. First, I describe mechanistic studies of the enzyme IspH, which is present in malaria parasites and most pathogenic bacteria, but not in humans. IspH is a 4Fe-4S protein and produces the five-carbon (C5) isoprenoids IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) from HMBPP (E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate) via a 2H(+)/2e(-) reduction (of an allyl alcohol to an alkene). The mechanism is unusual in that it involves organometallic species: "metallacycles" (η(2)-alkenes) and η(1)/η(3)-allyls. These observations lead to novel alkyne inhibitors, which also form metallacycles. Second, I describe structure-function-inhibition studies of FPP synthase, the macromolecule that condenses IPP and DMAPP to the sesquiterpene farnesyl diphosphate (FPP) in a "head-to-tail" manner. This enzyme uses a carbocation mechanism and is potently inhibited by bone resorption drugs (bisphosphonates), which I show are also antiparasitic agents that block sterol biosynthesis in protozoa. Moreover, "lipophilic" bisphosphonates inhibit protein prenylation and invasiveness in tumor cells, in addition to activating γδ T-cells to kill tumor cells, and are important new leads in oncology. Third, I describe structural and inhibition studies of a "head-to-head" triterpene synthase, dehydrosqualene synthase (CrtM), from Staphylococcus aureus. CrtM catalyzes the first committed step in biosynthesis of the carotenoid virulence factor staphyloxanthin: the condensation of two FPP molecules to produce a cyclopropane (presqualene diphosphate). The structure of CrtM is similar to that of human squalene synthase (SQS), and some SQS inhibitors (originally developed as cholesterol-lowering drugs) block staphyloxanthin biosynthesis. Treated bacteria are white and nonvirulent (because they lack the carotenoid shield that protects them from reactive oxygen species produced by neutrophils), rendering them susceptible to innate immune system clearance--a new therapeutic approach. And finally, I show that the heart drug amiodarone, also known to have antifungal activity, blocks ergosterol biosynthesis at the level of oxidosqualene cyclase in Trypanosoma cruzi, work that has led to its use in the clinic as a novel antiparasitic agent. In each of these four examples, I use information from one area (organometallic chemistry, bone resorption drugs, cholesterol-lowering agents, heart disease) to develop drug leads in an unrelated area: a "knowledge-based" approach that represents an important advance in the search for new drugs.

FIGURE 1

Structural results for IspH (LytB). A,B: Crystal structure results for Aquifex aeolicus IspH. C, Initial docking pose for HMBPP to oxidised IspH Fe₄S₄ cluster obtained by using the “open-form” structure. D, Comparison of HMBPP bound to IspH from X-ray (green) and docking (red). From Refs. , , with permission.

FIGURE 2

EPR and ENDOR results for IspH. A, EPR spectra of IspH (and an E126A mutant) ±ligands. B, ENDOR spectrum with [u-¹³C]-26. From Ref. , with permission.

FIGURE 3

IspH mechanism proposal. A, deoxygenation steps. B Reductive cleavage forming IPP, DMAPP from allyl species. From Ref. , with permission,

FIGURE 4

IspH inhibition by the alkyne diphosphate, 26. A, 9GHz ENDOR spectrum of [u-¹³C]-propargyl diphosphate (26) showing ~6 MHz ¹³C hyperfine coupling. B, Dose-response curve showing IspH inhibition by 26. C, docking results showing close apposition of the alkyne group to the unique, 4^th Fe in IspH. From Ref. , with permission.

FIGURE 5

Cationic bisphosphonates as FPPS/GGPPS inhibitors. A,B: φ(r) electrostatic potential surfaces for an ammonium diphosphate based terpene cyclase inhibitor (A)and ibandronate, B. C, Early model for bisphosphonate inhibition of FPPS. D, Crystal structure showing similar pose as in C. E, BPH-715 bound to GGPPS. From Refs. and , with permission.

FIGURE 6

Effects of the bisphosphonate pamidronate (29) on cutaneous Leishmaniasis (L. mexicana) in mice. A, effects of pamidronate dose on lesion progression. B, cure of infection in Treated mouse (is on the left). From Ref. , with permission.

FIGURE 7

CrtM as a target for anti-virulence therapy. A, comparison between CrtM (green) and SQS (yellow) structures. B, FSPP (two molecules) bound to CrtM. C, BPH-652 (40, in blue) bound to CrtM. The two FsPP molecules (green, yellow) are also shown. From Ref. with permission.

FIGURE 8

Effects of BPH-652 (40) on staphyloxanthin biosynthesis and S. aureus infection. A, BPH-652 blocks staphyloxanthin biosynthesis in cells. B, BPH-652 renders staph susceptible to killing by neutrophils in blood and C, reduces infectivity in mice by 98%. From Ref. , with permission.

Scheme I

Formation of Isopentenyl Diphosphate (1) and Dimethyallyl Diphosphate (2) in the Non-Mevalonate Pathway.

Scheme II

Formation of Farnesyl Diphosphate (6) and Geranylgeranyl Diphosphate (7)

Scheme III

Formation of Triterpenes from Farnesyl Diphosphate (6)

Scheme IV

Schematic Illustration of π/σ Bioorganometallic Species in Nitrogenase and IspH

Scheme V

Acetylene Inhibitors of IspH and Proposed Binding Mode

Scheme VI

Structures of Diphosphate, Several Bisphosphonates and a Terpene Cyclase Inhibitor

Scheme VII

Proposed Carbocation Mechanism for FPPS Catalysis and Similarity Between a Transition State/Reactive Intermediate and the Bisphosphonate Drug, Ibandronate

Scheme VIII

Some Inhibitors of the CrtM Enzyme from S. aureus

Scheme IX

Some Sterol Biosynthesis Inhibitors