Targeting the De Novo Purine Synthesis Pathway Through Adenylosuccinate Lyase Depletion Impairs Liver Cancer Growth by Perturbing Mitochondrial Function.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. APPROACH AND RESULTS: We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. CONCLUSIONS: Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.

Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design.

There is significant clinical need for new antifungal agents to manage infections with pathogenic species such as Cryptococcus neoformans Because the purine biosynthesis pathway is essential for many metabolic processes, such as synthesis of DNA and RNA and energy generation, it may represent a potential target for developing new antifungals. Within this pathway, the bifunctional enzyme adenylosuccinate (ADS) lyase plays a role in the formation of the key intermediates inosine monophosphate and AMP involved in the synthesis of ATP and GTP, prompting us to investigate ADS lyase in C. neoformans. Here, we report that ADE13 encodes ADS lyase in C. neoformans. We found that an ade13Δ mutant is an adenine auxotroph and is unable to successfully cause infections in a murine model of virulence. Plate assays revealed that production of a number of virulence factors essential for dissemination and survival of C. neoformans in a host environment was compromised even with the addition of exogenous adenine. Purified recombinant C. neoformans ADS lyase shows catalytic activity similar to its human counterpart, and its crystal structure, the first fungal ADS lyase structure determined, shows a high degree of structural similarity to that of human ADS lyase. Two potentially important amino acid differences are identified in the C. neoformans crystal structure, in particular a threonine residue that may serve as an additional point of binding for a fungal enzyme-specific inhibitor. Besides serving as an antimicrobial target, C. neoformans ADS lyase inhibitors may also serve as potential therapeutics for metabolic disease; rather than disrupt ADS lyase, compounds that improve the stability the enzyme may be used to treat ADS lyase deficiency disease.

Structure of Staphylococcus aureus adenylosuccinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery.

The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enzyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit-subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.

Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate lyase produces skeletal muscle dysfunction.

Controversy exists as to whether the purine nucleotide cycle is important in normal skeletal muscle function. Patients with disruption of the cycle from a deficiency of AMP deaminase exhibit variable degrees of muscle dysfunction. An animal model was used to examine the effect of inhibition of the purine nucleotide cycle on muscle function. When the compound 5-amino-4-imidazolecarboxamide riboside (AICAriboside) is phosphorylated to the riboside monophosphate in the myocyte it is an inhibitor of adenylosuccinate lyase, one of the enzymes of the purine nucleotide cycle. AICAriboside was infused in 28 mice, and 22 mice received saline. Gastrocnemius muscle function was assessed in situ by recording isometric tension developed during stimulation. The purine nucleotide content of the muscle was measured before and after stimulation. Disruption of the purine nucleotide cycle during muscle stimulation was evidenced by a greater accumulation of adenylosuccinate, the substrate for adenylosuccinate lyase, in the animals receiving AICAriboside (0.60 +/- 0.10 vs. 0.05 +/- 0.01 nmol/mumol total creatine, P less than 0.0001). There was also a larger accumulation of inosine monophosphate in the AICAriboside vs. saline-treated animals at end stimulation (73 +/- 6 vs. 56 +/- 5 nmol/mumol total creatine, P less than 0.03). Inhibition of flux through the cycle was accompanied by muscle dysfunction during stimulation. Total developed tension in the AICAriboside group was 40% less than in the saline group (3,023 +/- 1,170 vs. 5,090 +/- 450 g . s, P less than 0.002). An index of energy production can be obtained by comparing the change in total phosphagen content per unit of developed tension in the two groups. This index indicates that less high energy phosphate compounds were generated in the AICAriboside group, suggesting that interruption of the purine nucleotide cycle interfered with energy production in the muscle. We conclude from these studies that defective energy generation is one mechanism whereby disruption of the purine nucleotide cycle produces muscle dysfunction.

Succinylpurinemic autism: increased sensitivity of defective adenylosuccinate lyase towards 4-hydroxy-2-nonenal.

We studied the effect of trans-4-hydroxy-2-nonenal on the wild-type human adenylosuccinate lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10-50 microM trans-4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans-2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans-4-hydroxy-2-nonenal on both the wild-type and the defective adenylosuccinate lyase, while it did not produce practically any change in the presence of trans-2,3-nonenal or nonanal.

Inhibition of defective adenylosuccinate lyase by HNE: a neurological disease that may be affected by oxidative stress.

Adenylosuccinate lyase is an enzyme of fumarase superfamily that participates in the purine biosynthetic pathway, catalysing the nonhydrolytic cleavage of succinyl groups from SAICA ribotide and adenylosuccinate. Enzyme defects are associated with a human inherited disease, which arises from single point mutations to the gene and results in mild to severe psychomotor retardation, epilepsy, muscle wasting, and autistic features. Adenylosuccinate lyase activity is lost to a different extent in the patients. Diminished levels of enzyme have been attributed to loss of catalytic activity, protein instability, or environmental factors. P100A/D422Y mutation represents a feasible model for studying the effect of cell milieu on the activity of the impaired enzyme. The defective enzyme is inhibited by micromolar concentrations of trans-4-hydroxy-2-nonenal (HNE), a major product of membrane peroxidation that has been found to accumulate in brain tissues of patients with neurodegenerative disorders. It is suggested that inactivation of defective adenylosuccinate lyase by HNE and other membrane peroxidation products may account, at least in part, for the impairment of neurological functions and recurrent worsening of the symptoms.

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet ß cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human ß cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in ß cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing ß cell dysfunction in T2D.

Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR).

L-Alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR) has been synthesized enzymatically using 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase in conjunction with 5-aminoimidazole-4-carboxylic acid ribonucleotide and L-2-amino-3-(N-hydroxy-N-nitrosoamino)propionic acid (alanosine). The product was characterized by chromatography, ultraviolet spectrum and NMR spectrum at 300 MHz. Alanosyl-AICOR was not a substrate of adenylosuccinate lyase from rat skeletal muscle, but it was an apparent competitive inhibitor in both of the reactions catalyzed by the enzyme. The KI values for alanosyl-AICOR were approximately 1.5 and 1.3 microM in the SAICAR and adenylosuccinate cleavage reactions respectively. These KI values were essentially the same as the Km values for the two substrates of adenylosuccinate lyase. They compare with an accumulation of 70 microM alanosyl-AICOR in leukemic nodules of mice treated with alanosine [A. K. Tyagi and D. Cooney, Cancer Res. 40, 4390 (1980)]. Thus, inhibition of adenylosuccinate lyase may account for much of the inhibitory effect exerted by alanosyl-AICOR in vivo. We confirmed the previous observation that alanosyl-AICOR is an inhibitor of adenylosuccinate synthetase.

Transition-state inhibitors of purine salvage and other prospective enzyme targets in malaria.

Malaria is a leading cause of human death within the tropics. The gradual generation of drug resistance imposes an urgent need for the development of new and selective antimalarial agents. Kinetic isotope effects coupled to computational chemistry have provided the relevant details on geometry and charge of enzymatic transition states to facilitate the design of transition-state analogs. These features have been reproduced into chemically stable mimics through synthetic chemistry, generating inhibitors with dissociation constants in the pico- to femto-molar range. Transition-state analogs are expected to contribute to the control of malaria.

The interaction of monofluorofumarate with adenylosuccinate lyase.

Monofluorofumarate was tested as an alternate substrate and inhibitor for adenylosuccinate lyase. Monofluorofumarate was found to be a slow reacting substrate when either AMP or AICAR (5-aminoimidazole 4-carboxamide ribonucleotide) were used as substrate acceptor molecules at pH 7.5. There was no indication that monofluorofumarate could induce the inactivation of adenylosuccinate lyase. The initial reaction product when monofluorofumarate was incubated with AMP in the presence of adenylosuccinate lyase has been determined to be 2-fluoro-adenylosuccinate. This molecule lost HF spontaneously, and the subsequent intermediate was rapidly hydrolyzed to oxalacetate and AMP. A similar reaction scheme was also observed when AICAR was utilized as a cosubstrate with monofluorofumarate. The initial reaction rate when 1.0 mM monofluorofumarate and 1.0 mM AMP were used as substrates with adenylosuccinate lyase was only 1.4% of the rate when 1.0 mM fumarate was used. AICAR (1.0 mM) was found to react with monofluorofumarate at 8.9% of the rate that it reacts with fumarate.

Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at -70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

Affinity Labeling of the active site of rabbit muscle adenylosuccinate lyase by 2-[(4-bromo-2.3-dioxobutyl)thio] adenosine 5'-monophosphate.

Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 muM 2 -[(4-bromo-2.3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, ph 7.0 and 10 degrees C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (K1=8.5 microM. 5.2 microM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB-[14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5'-AMP and by 0.1 mM 5'-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5'-AMP alone, 5 mM fumarate alone, or 0.1 mM 5'-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB-TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase.

Metabolism of threo-beta-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates.

DL-threo-beta-Fluoroaspartate is a substrate for the two enzymes in de novo purine biosynthesis that use aspartate, namely 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase and adenylosuccinate synthetase. With both enzymes, Vmax with threo-beta-fluoroaspartate is about 50% of that observed with aspartate. The products of the two enzyme reactions, threo-beta-fluoro-SAICAR and threo-beta-fluoroadenylosuccinate, are inhibitors of adenylosuccinate lyase purified from rat skeletal muscle. In 20 mM phosphate buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are 5 and 3 microM and for threo-beta-fluoroadenylosuccinate are 3 and 1 microM, in the SAICAR and adenylosuccinate cleavage reactions, respectively. In 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are approximately 0.14 and 0.03 microM and for threo-beta-fluoroadenylosuccinate are approximately 0.05 and 0.015 microM, in the same two reactions, respectively. These KI values are one-half to one-hundredth of the Km values for SAICAR and adenylosuccinate, the two substrates of adenylosuccinate lyase. After an 8-h incubation with 45 microM threo-beta-fluoroaspartate, H4 cells contain 200-300 microM threo-beta-fluoro-SAICAR and 60-90 microM threo-beta-fluoroadenylosuccinate. These concentrations of fluoro analogs are sufficient to substantially inhibit adenylosuccinate lyase and hence the de novo synthesis of purines in H4 cells.