Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

Kinetic and structural studies of Trypanosoma and Leishmania phosphofructokinases show evolutionary divergence and identify AMP as a switch regulating glycolysis versus gluconeogenesis.

Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK - to a much larger extent than Trypanosoma PFK - is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10-fold, compared to only a 1.3- and 1.4-fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme's 'reaching arms' and 'embracing arms' that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T-to-R equilibria for the two families, with the low-melting Leishmania PFK favouring the flexible inactive T-state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP-dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.

The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction.

Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro, allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate.

Structures of Leishmania Fructose-1,6-Bisphosphatase Reveal Species-Specific Differences in the Mechanism of Allosteric Inhibition.

The gluconeogenic enzyme fructose-1,6-bisphosphatase has been proposed as a potential drug target against Leishmania parasites that cause up to 20,000-30,000 deaths annually. A comparison of three crystal structures of Leishmania major fructose-1,6-bisphosphatase (LmFBPase) along with enzyme kinetic data show how AMP acts as an allosteric inhibitor and provides insight into its metal-dependent reaction mechanism. The crystal structure of the apoenzyme form of LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered "dynamic loops". The structure of LmFBPase, complexed with manganese and its catalytic product phosphate, shows the dynamic loops locked into the active sites. A third crystal structure of LmFBPase complexed with its allosteric inhibitor AMP shows an inactive form of the tetramer, in which the dimer pairs are rotated by 18° relative to each other. The three structures suggest an allosteric mechanism in which AMP binding triggers a rearrangement of hydrogen bonds across the large and small interfaces. Retraction of the "effector loop" required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state. The structure of the effector site of LmFBPase shows different structural features compared with human FBPases, thereby offering a potential and species-specific drug target.

Pushing the Limits of Detection of Weak Binding Using Fragment-Based Drug Discovery: Identification of New Cyclophilin Binders.

Fragment-based drug discovery is an increasingly popular method to identify novel small-molecule drug candidates. One of the limitations of the approach is the difficulty of accurately characterizing weak binding events. This work reports a combination of X-ray diffraction, surface plasmon resonance experiments and molecular dynamics simulations for the characterization of binders to different isoforms of the cyclophilin (Cyp) protein family. Although several Cyp inhibitors have been reported in the literature, it has proven challenging to achieve high binding selectivity for different isoforms of this protein family. The present studies have led to the identification of several structurally novel fragments that bind to diverse Cyp isoforms in distinct pockets with low millimolar dissociation constants. A detailed comparison of the merits and drawbacks of the experimental and computational techniques is presented, and emerging strategies for designing ligands with enhanced isoform specificity are described.

Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new nonpeptide inhibitor using Biacore™ T200.

We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A. Our orientation-specific stabilisation approach captures his-tagged protein under 'physiological conditions' (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni2+-nitrilotriacetic acid surfaces, very briefly activated for primary amine-coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin-A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin-A-cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin-A, from a screen of a fragment library. This fragment, 2,3-diaminopyridine, bound specifically with a mean affinity of 248 ± 60 µm. The X-ray structure of this 109-Da fragment bound in the active site of cyclophilin-A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin-A inhibitors.

A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM.

We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors.

Identification of ML251, a Potent Inhibitor of T. brucei and T. cruzi Phosphofructokinase.

Human African Trypanosomiasis (HAT) is a severe, often fatal disease caused by the parasitic protist Trypanosoma brucei. The glycolytic pathway has been identified as the sole mechanism for ATP generation in the infective stage of these organisms, and several glycolytic enzymes, phosphofructokinase (PFK) in particular, have shown promise as potential drug targets. Herein, we describe the discovery of ML251, a novel nanomolar inhibitor of T. brucei PFK, and the structure-activity relationships within the series.

Pyruvate kinases have an intrinsic and conserved decarboxylase activity.

The phosphotransfer mechanism of PYKs (pyruvate kinases) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in the present study to follow OAA (oxaloacetate) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of TbPYK (Trypanosoma brucei PYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, 2-oxoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK has a relatively low turnover with values of 0.86 s-1 and 1.47 s-1 in the absence and presence of F26BP (fructose 2,6-bisphosphate) respectively. Human M1PYK (M1 isoform of PYK) has a measured turnover value of 0.50 s-1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-oxo acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilized enol intermediate.

Structures of pyruvate kinases display evolutionarily divergent allosteric strategies.

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k cat/S 0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k cat/S 0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.

The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

11ß-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo.

11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7ß-hydroxy- and 7-keto-cholesterol (7ßOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11ßHSD1 to the balance of circulating levels of 7KC and 7ßOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7ßOHC in mice were 91.3±22.3 and 22.6±5.7 nM respectively, increasing to 1240±220 and 406±39 nM in ApoE(-/-) mice receiving atherogenic western diet. Disruption of 11ßHSD1 in mice increased (p<0.05) the 7KC/7ßOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7ßOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11ßHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7ßOHC was a competitive inhibitor of oxidation of corticosterone (Ki=0.9 µM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11ßHSD1 influenced the abundance and balance of circulating and tissue levels of 7ßOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors.

A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

Bivalent enzyme inhibitors discovered using dynamic covalent chemistry.

A bivalent dynamic covalent chemistry (DCC) system has been designed to selectively target members of the homodimeric glutathione-S-transferase (GST) enzyme family. The dynamic covalent libraries (DCLs) use aniline-catalysed acylhydrazone exchange between bivalent hydrazides and glutathione-conjugated aldehydes and the bis-hydrazides act as linkers to bridge between each glutathione binding site. The resultant DCLs were found to be compatible and highly responsive to templating with different GST isozymes, with the best results coming from the M and Schistosoma japonicum (Sj) class of GSTs, targets in cancer and tropical disease, respectively. The approach yielded compounds with selective, nanomolar affinity (K(i) =61 nM for mGSTM1-1) and demonstrates that DCC can be used to simultaneously interrogate binding sites on different subunits of a dimeric protein.

Phosphoglycerate mutase from Trypanosoma brucei is hyperactivated by cobalt in vitro, but not in vivo.

Production of ATP by the glycolytic pathway in the mammalian pathogenic stage of protists from the genus Trypanosoma is required for the survival of the parasites. Cofactor-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shows no similarity to the corresponding enzyme in humans, and has also been genetically validated as a target by RNAi experiments. It has previously been shown that trypanosomatid iPGAMs require Co(2+) to reach maximal activity, but the biologically relevant metal has remained unclear. In this paper the metal content in the cytosol of procyclic and bloodstream-form T. brucei (analysed by inductively coupled plasma-optical emission spectroscopy) shows that Mg(2+), Zn(2+) and Fe(2+) were the most abundant, whereas Co(2+) was below the limit of detection (<0.035 µM). The low concentration indicates that Co(2+) is unlikely to be the biologically relevant metal, but that instead, Mg(2+) and/or Zn(2+) may assume this role. Results from metal analysis of purified Leishmania mexicana iPGAM by inductively coupled plasma-mass spectrometry also show high concentrations of Mg(2+) and Zn(2+), and are consistent with this proposal. Our data suggest that in vivo cellular conditions lacking Co(2+) are unable to support the maximal activity of iPGAM, but instead maintain its activity at a relatively low level by using Mg(2+) and/or Zn(2+). The physiological significance of these observations is being pursued by structural, biochemical and biophysical studies.

The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site.

Ehrlich's pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye molecules including trypan blue and trypan red to eliminate trypanosome infections in mice. The molecular structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clinical use. Despite the biological importance of these dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, K(i) = 1.1-17 µM), T. cruzi (K(i) = 108 µM), and L. mexicana (K(i) = 116 µM), all of which have similar active sites. Synergistic effects were observed when examining suramin inhibition in the presence of an allosteric effector molecule, whereby IC(50) values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for suramin and into the mode of action of the dye-like molecules used in Ehrlich's original experiments.

Pyrazolo[4,3-d]pyrimidine bioisostere of roscovitine: evaluation of a novel selective inhibitor of cyclin-dependent kinases with antiproliferative activity.

Inhibition of cyclin-dependent kinases (CDKs) with small molecules has been suggested as a strategy for treatment of cancer, based on deregulation of CDKs commonly found in many types of human tumors. Here, a new potent CDK2 inhibitor with pyrazolo[4,3-d]pyrimidine scaffold has been synthesized, characterized, and evaluated in cellular and biochemical assays. 7-Benzylamino-5(R)-[2-(hydroxymethyl)propyl]amino-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine, compound 7, was prepared as a bioisostere of the well-known CDK inhibitor roscovitine. An X-ray crystal structure of compound 7 bound to CDK2 has been determined, revealing a binding mode similar to that of roscovitine. Protein kinase selectivity profile of compound 7 and its biological effects (cell cycle arrest, dephosphorylation of the retinoblastoma protein, accumulation of the tumor suppressor protein p53, induction of apoptosis, inhibition of homologous recombination) are consistent with CDK inhibition as a primary mode of action. Importantly, as the anticancer activities of the pyrazolo[4,3-d]pyrimidine 7 exceed those of its bioisostere roscovitine, compound 7 reported here may be preferable for cancer therapy.