The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

The relationship between the chemistry and biological activity of the bisphosphonates.

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.

Farnesyl pyrophosphate synthase enantiospecificity with a chiral risedronate analog, [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501): Synthetic, structural, and modeling studies.

The complex formed from crystallization of human farnesyl pyrophosphate synthase (hFPPS) from a solution of racemic [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501, 8), a chiral analog of the anti-osteoporotic drug risedronate, contained the R enantiomer in the enzyme active site. This enantiospecificity was assessed by computer modeling of inhibitor-active site interactions using Autodock 3, which was also evaluated for predictive ability in calculations of the known configurations of risedronate, zoledronate, and minodronate complexed in the active site of hFPPS. In comparison with these structures, the 8 complex exhibited certain differences, including the presence of only one Mg(2+), which could contribute to its 100-fold higher IC(50). An improved synthesis of 8 is described, which decreases the number of steps from 12 to 8 and increases the overall yield by 17-fold.

Structure-activity relationships among the nitrogen containing bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl pyrophosphate synthase.

The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.

Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy.

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.