Small-molecule inhibitors of cathepsin L incorporating functionalized ring-fused molecular frameworks

Cathepsin L is a cysteine protease that is upregulated in a variety of malignant tumors and plays a significant role in cancer cell invasion and migration. It is an attractive target for the development of small-molecule inhibitors, which may prove beneficial as treatment agents to limit or arrest cancer metastasis. We have previously identified a structurally diverse series of thiosemicarbazone-based inhibitors that incorporate the benzophenone and thiochromanone molecular scaffolds. Herein we report an important extension of this work designed to explore fused aryl–alkyl ring molecular systems that feature nitrogen atom incorporation (dihydroquinoline-based) and carbon atom exclusivity (tetrahydronaphthalene-based). In addition, analogues that contain oxygen (chromanone-based), sulfur (thiochroman-based), sulfoxide, and sulfone functionalization have been prepared in order to further investigate the structure–activity relationship aspects associated with these compounds and their ability to inhibit cathepsins L and B. From this small-library of 30 compounds, five were found to be strongly inhibitory (IC₅₀ <500 nM) against cathepsin L with the most active compound (7-bromodihydroquinoline thiosemicarbazone 48) demonstrating an IC₅₀ = 164 nM. All of the compounds evaluated were inactive (IC₅₀ >10,000 nM) as inhibitors of cathepsin B, thus establishing a high degree (>20-fold) of selectivity (cathepsin L vs. cathepsin B) for the most active cathepsin L inhibitors in this series.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

Design,synthesis, and biological evaluation of potent thiosemicarbazone based cathepsin L inhibitors

A small library of 36 functionalized benzophenone thiosemicarbazone analogs has been prepared by chemical synthesis and evaluated for their ability to inhibit the cysteine proteases cathepsin L and cathepsin B. Inhibitors of cathepsins L and B have the potential to limit or arrest cancer metastasis. The six most active inhibitors of cathepsin L (IC₅₀ < 85 nM) in this series incorporate a meta-bromo substituent in one aryl ring along with a variety of functional groups in the second aryl ring. These six analogs are selective for their inhibition of cathepsin L versus cathepsin B (IC₅₀ > 10,000 nM). The most active analog in the series, 3-bromophenyl-2′-fluorophenyl thiosemicarbazone 1, also efficiently inhibits cell invasion of the DU-145 human prostate cancer cell line.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K_m and V_max values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K_m values at optimum pH and 20°C were 0.625?mM, 0.541?mM, 0.785?mM and 0.862?mM with p-nitro phenyl acetate, for all CA localizations. The respective V_max values at optimum pH and 20°C were 0.875?μmol/L?min, 0.186?μmol/L?min, 0.214?μmol/L?min and 0.253?μmol/L?min with the same substrate. The K_i and I₅₀ values for the inhibitors sulphanilamide, KSCN, NaN₃ and acetazolamide were determined for all the CA localizations.     

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design,synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (K_i = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (K_i = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.     

Synthesis,carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines

4-(3-(4-Substituted-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamides (9–16) were successfully synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR, and HRMS spectra. Carbonic anhydrase I and II inhibitory effects of the compounds were investigated. K_i values of the compounds were in the range of 316.7?±?9.6–533.1?±?187.8?nM towards hCA I and 412.5?±?115.4–624.6?±?168.2?nM towards hCA II isoenzymes. While K_i values of the reference compound Acetazolamide were 278.8?±?44.3?nM and 293.4?±?46.4?nM towards hCA I and hCA II izoenzymes, respectively. Compound 14 with bromine and compound 13 with fluorine substituents can be considered as the leader compounds of the series because of the lowest K_i values in series to make further detailed carbonic anhydrase inhibiton studies.     

Optimization of Mixed Cultivation of the Moderate Thermophilic Bioleaching Microorganisms for High Cell Density Using Statistical Methodology

The low functional microbial population density in the industrial bioleaching process has been a limiting factor for the high leaching efficiency, making the microbial cultivation and continuous inoculation an alternative for sustaining the microbial activity. In the present experiment, the defined mixed cultivation of Leptospirillum ferriphilum YSK, Sulfobacillus acidophilus TPY, Acidithiobacillus caldus S2, and Ferroplasma thermophilum L1 was evaluated and optimized by Statistical Methodology. Going through the Plackett–Burman experimental design, pH value, temperature, and c(MgSO₄·7H₂O) were considered as the most significant factors in the defined range. Then, the relationships were analyzed using the steepest ascent design, the central composite design, and finally the response surface methodology. It was suggested that the optimum parameters were pH 1.38, MgSO₄·7H₂O 0.552?g/L, temperature 44?°C, FeSO₄·7H₂O 40?g/L, sulfur 8?g/L, yeast 0.02% w/v, (NH₄)₂SO₄ 3g/L, K₂HPO₄ 0.5g/L, KCl 0.1g/L, Ca(NO₃)₂ 0.01?g/L, in which allowed total cell density of the microbial community to reach 7.63?×?10⁸ cells/mL in the cultivation period. The lab experiments were routinely undertaken with the expectation that the L. ferriphilum YSK, S. acidophilus TPY, A. caldus S2, F. thermophilum L1 could rapid grown from initial cell density of 0.25?×?10⁷ cells/mL to 2.82?×?10⁸ cells/mL, 1.68?×?10⁸ cells/mL, 2.76?×?10⁸ cells/mL, 2.51?×?10⁷ cells/mL, respectively in 58?h. It demonstrates a possibility to co-culture these microbes in a single reactor, providing an efficient way to regenerate of inoculation for biomining process.     

Synthesis and biological evaluation of a water-soluble phosphate prodrug salt and structural analogues of KGP94, a lead inhibitor of cathepsin L

The magnitude of expression of cathepsin L, often upregulated in the tumor microenvironment, correlates with the invasive and metastatic nature of certain tumors. Inhibition of cathepsin L represents an emerging strategy for the treatment of metastatic cancer. A potent, small-molecule inhibitor (referred to as KGP94) of cathepsin L, and new KGP94 analogues were synthesized. (3,5-Dibromophenyl)-(3-hydroxyphenyl) ketone thiosemicarbazone (22), with an IC₅₀ value of 202 nM, exhibited similar inhibitory activity against cathepsin L compared to KGP94 (IC₅₀ = 189 nM). Due to limited aqueous solubility of KGP94, a water-soluble phosphate salt (KGP420) was prepared in order to facilitate future in vivo studies. Enzymatic hydrolysis with alkaline phosphatase (ALP) demonstrated that the phosphate prodrug, KGP420, was readily converted to the parent compound, KGP94.     

Electric dichroism studies on ferriheme– and ferroheme–poly(L-lysine) complexes at pH 9–12

Transient electric dichroism has been measured for the ferriheme–poly(L -lysine)[(Lys)_n], ferroheme–(Lys)_n, and ferroheme–(Lys)_n–carbon monoxide (CO) solutions at pH 9–12. The Soret absorption maximum in electronic spectrum (λ), the reduced linear dichroism (ρ_∞) at complete orientation and the calculated angle (?) between the porphyrin plane of a bound heme and the oriented polymer axis are determined for the following complexes: a ferriheme–(Lys)_n complex at pH 9.5–10.5 (λ = 420 nm, ρ_∞ = 0.50, and ? = 19°), a ferroheme–(Lys)_n complex at pH 9.5–10.2 (λ = 432 nm, ρ_∞ = 0.77, and ? = 0°), and a ferroheme–(Lys)_n–CO complex at pH 9.25 (λ = 430 nm, ρ_∞ = 0.38, and ? = 24°). Based on the above data, the validity of the structures of heme–(Lys)_n complexes proposed by other investigators is discussed.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.     

Design,synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC₅₀ values and inhibition constants (K_i) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with K_i amounting to 1.14 and 7.21?nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5?µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1?µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Dynamic light-scattering studies of internal motions in DNA. III. Evidence for titratable joints associated with bound polycations

Dynamic light-scattering techniques are employed to study the internal Brownian motions of a commercial calf thymus DNA, clean and contaminated ?29 DNAs, and a clean ?29 DNA with bound spermidine as a function of pH. The Rouse-Zimm model parameters of both calf thymus and contaminated ?29 DNAs differ substantially from those of clean ?29 DNA in the neutral-pH region. However, this difference is largely removed by adding 0.01M EDTA (which has no effect on clean ?29 DNA) to the calf thymus DNA sample. These findings imply the existence in that preparation of polycation contaminants, presumably basic proteins, that can substantially alter the local mechanical properties of the DNA near their binding sites. The internal motion parameters k_BT/f and b of both calf thymus and contaminated ?29 DNAs are found to exhibit pronounced characteristic variations between pH 8.5 and 10.5, over which range there is essentially no detectable titration to a resolution of about 1% of the base pairs. These variations, which are not observed for clean ?29 DNA, are qualitatively similar to those previously reported for a ?29 DNA with 21 single-strand breaks per chain. This indicates the formation of titratable joints associated with bound polycation contaminants. These basic ligands presumably facilitate local denaturation by stabilizing the titration of one or more protons on base-ring nitrogens near their binding sites. Spermidine binding up to 85–87% of neutralization of the total DNA charge has only a relatively minor effect on the internal motion parameters at neutral pH in 0.01M NaCl. However on raising the pH to 10.2, the internal motion parameter k_BT/f undergoes a marked decrease similar to that observed for both calf thymus and contaminated ?29 DNAs and also ?29 DNA with single-strand breaks. This indicates that spermidine, too, is capable of inducing titratable joints. Evidence is presented that the titratable joints associated with bound polycations on the calf thymus DNA may serve primarily as torsion joints, as was found previously for the titratable joints associated with single-strand breaks.     

Solid state fermentation of Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor for tannase production

Abstract

Production of tannase was performed in packed bed reactor filled with an inert support polyurethane foam (PUF) using Bacillus gottheilii M2S2. The influence of process parameters such as fermentation time (24–72?h), tannic acid concentration (0.5–2.5% w/v), inoculum size (7–12% v/v), and aeration rate (0–0.2?L/min) on tannase production with PUF were analyzed using one variable at a time (OVAT) approach. The outcome of OVAT was optimized by central composite design. Based on the statistical investigation, the proposed mathematical model recommends 1% (w/v) of tannic acid, 10% (v/v) of inoculum size and 0.13?L/min of aeration rate for maximum production (76.57?±?1.25?U/L). The crude enzyme was purified using ammonium sulfate salt precipitation method followed by dialysis. The biochemical properties of partially purified tannase were analyzed and found the optimum pH (4.0), temperature (40?°C) for activity, and K_m (1.077?mM) and V_max (1.11?µM/min) with methyl gallate as a substrate. Based on the SDS-PAGE analysis, tannase exhibited two bands with molecular weights of 57.5 and 42.3?kDa. Briefly, the partially purified tannase showed 4.2 fold increase (63?±?1.60?U/L) in comparison to the submerged fermentation and the production of tannase was validated by using NMR spectrometer.     

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: Kinetic characterization

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (AChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration-dependent manner; IC₅₀ was found to be 8.07 nM. The Michaelis-Menten constant (K_a) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54–67% in the tacrine-treated systems. The V_max was 0.701 μ mole/min per milligram protein for the control system, but it was decreased by 26–69% in the tacrine-treated systems. The Lineweaver–Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of K_i and K_t were estimated to be as 4.475 and 8.517 nM, respectively. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 245–251, 1998     

Inhibitory Effect of di- and Tripeptidyl Aldehydes on Calpains and Cathepsins

Abstract

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of α-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B < calpain II ≈ calpain I < cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (K_i) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (K_i, 36 nM) and calpain II (K_i, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (K_i, 0.5nM); acetyl-Leu-Leu-Met-H for cathepsin B (K_i, 100nM).     

Fumigant toxicity of Laurus nobilis and Myrtus communis essential oils on larvae and adults of the Red flour beetle,Tribolium castaneum Herbst (Col.: Tenebrionidae)

Considering the invasion to food commodities by insects and harmful effect of chemical pesticides, essential oils are among the best known substances tested against stored product pests. These compounds may act as fumigants, contact insecticides, repellents or anti-feedants. In present study, fumigant toxicity of essential oils from Laurus nobilis L. and Myrtus communis L. was assessed on larvae and adults of Tribolium castaneum Herbst at 27?±?2?°C, 60?±?5% RH in darkness. Each essential oil was tested in five concentrations with three replicates. The LC₅₀ values of L. nobilis and M. communis against adults of beetle were calculated 243.78 and 56.11?μl/l and LC₉₅ values for them were 685.85 and 144.01?μl/l, respectively. For the larvae of T. castaneum, the LC₅₀ values for L. nobilis and M. communis were 211.64 and 69.63 and LC₉₅ values were 656.84 and 183.65?μl/l, respectively. Results showed that these essential oils may have potential as botanical control agents against larvae and adults of T. castaneum.     

Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I,II and Mycobacterium tuberculosis β-class enzyme Rv3273

A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, ¹H- and ¹³C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The K_Is were in the range of 54.6?nM–1.8?µM against hCA I, in the range of 32.1?nM–5.5?µM against hCA II and of 127?nM–2.12?µM against mtCA 3.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Pyrrolo[3,2-c]pyridine derivatives with potential inhibitory effect against FMS kinase: in vitro biological studies

A series of eighteen pyrrolo[3,2-c]pyridine derivatives were tested for inhibitory effect against FMS kinase. Compounds 1e and 1r were the most potent among all the other tested analogues (IC₅₀?=?60?nM and 30?nM, respectively). They were 1.6 and 3.2 times, respectively, more potent than our lead compound, KIST101029 (IC₅₀?=?96?nM). Compound 1r was tested over a panel of 40 kinases including FMS, and exerted selectivity against FMS kinase. It was further tested against bone marrow-derived macrophages (BMDM) and its IC₅₀ was 84?nM (2.32-fold more potent than KIST101029 (IC₅₀?=?195?nM)). Compound 1r was also tested for antiproliferative activity against a panel of six ovarian, two prostate, and five breast cancer cell lines, and its IC₅₀ values ranged from 0.15–1.78?µM. It possesses also the merit of selectivity towards cancer cells than normal fibroblasts.