Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

On the possible influence of different pathways of binding of amides with water on “Pyramidalization” of valence bonds of peptide group nitrogen

With the aim to study solvation effects in peptide structure organization, the behavior of the energy of different types of hydration in simple amines and amides has been analyzed. On the example of quantum-chemical DFT and PM3 calculations of amino derivatives of composition CH₃-(CH₂)₃)-NH₂, (CH3)₂-NH, CH₃-NH₂, NH₃, CH₂=CH-NH₂, H-CC-NH₂, O=C(CH)₃-N(CH₃)₂, O=C(CH₃)-NH(CH₃), O=C(CH₃)-NH₂, O=CH-N(CH₃)H, and O=CH-NH₂ it has been shown that: (1) in the given set of molecules, the proton acceptor N…H-O variant of hydrogen bonding of NH₂ group with a water molecule is dominating only for the simplest amines. Being primordially weaker, the proton donor N-H…OH variant of water H-bonding gradually increases in energy in the given set as the basicity of the compound decreases, and for the case of amides of carboxylic acids it becomes already a significant channel of the hydration; (2) the intermolecular N-H…O=C bonding of trans-N-methylacetamides, which models the peptide hydrogen bonds in proteins, induces “planarization” of its initially nonplanar O=C-NH fragments. However, the addition of water molecules to the complex through the proton acceptor N…H-O variant of binding of N atom not only restores but even strengthens the “pyramidalization” of valence bonds of peptide groups.     

Single and double nucleophilic attack on cluster-bonded allenylidenes: Synthesis of phosphorus heterocycles

Further studies of the attack of bidentate tertiary phosphines, such as dppm and dppe, and the related diarsine dpam, on Ru₃(μ-H){μ₃-C₂CHR(OH)}(CO)₉ (R = H, Me) are described, together with the X-ray determined structures of [Ru₃(μ₃-C₂CH₂PPh₂CH₂CH₂PPh₂)(CO)₉]BF₄ (6) and Ru₃(μ-H){μ₃-C₂CHMePPh₂CHPPh₂}(CO)₉ (7) (containing diphospha heterocycles), Ru₃(μ-H){μ₃-CH₂(OH)C₂PPh₂CH₂PPh₂}(CO)₈ (8) (containing a phosphonium-alkynyl ligand also coordinated to a cluster Ru atom) and Ru₃(μ-H)(μ₃-CCCHAsPh₂CH₂AsPh₂)(CO)₉ (9) (containing an arsino-allenylidene ligand). These complexes are formed by nucleophilic attack of the Group 15 ligand on an alkynyl or allenylidene ligand on the cluster.     

Novel synthetic 2-amino-10-(3,5-dimethoxy)benzyl-9(10H)-acridinone derivatives as potent DNA-binding antiproliferative agents

A series of novel 9(10H)-acridinone derivatives with terminal amino substituents at C2 position on the acridinone ring were synthesized and studied for their antiproliferative activity and underlying mechanisms. These compounds demonstrated promising cytotoxicity to leukemia cells CCRF-CEM, displaying IC₅₀ values in the low micromolar range. Structure–activity relationships (SAR) indicated that the compound 6d bearing a pyrrolidine substituent and 8a with a methyl ammonium side chain displayed higher cytotoxicity to CCRF-CEM cells and also solid tumor cells A549, HepG2, and MCF7. Furthermore, the compounds 6d and 8a had strong binding activity to calf thymus DNA (ct DNA), as detected by UV absorption and fluorescence quenching assays, but limited inhibitory activity to human topoisomerase 1 (topo 1). Taken together, this study discovered a series of new synthetic 9(10H)-acridinone derivatives with potent DNA binding and anticancer activity.     

Kinetics and mechanism of the oxidative addition of iodomethane to β-diketonatobis(triphenylphosphite)rhodium(I) complexes

The kinetics and mechanism of the oxidative addition of CH₃I to [Rh(β-diketone)(P(OPh)₃)₂] complexes was studied in acetone medium at various temperatures. The experimental rate law is R = k[Rh(β-diketone)(P(OPh₃)₂][CH₃I]. The order of the effect of the β-diketone on the reactivity of the complexes is acac >; BA >; DBM >; TFAA >; TFBA >; HFAA indicating that electronegative substituents of the β-diketone decrease the reactivity of the complexes towards oxidative addition reactions. The volume of activation for some of the reactions was determined in various solvents. The large negative values of the volume and entropy of the activation indicated a mechanism which occurs via a polar transition state.     

Syntheses and structural analyses of chiral rhenium containing amines of the formula (η-C5H5)Re(NO)(PPh3)((CH2)nNRR′) (n = 0, 1)

The reaction of the racemic chiral methyl complex (η⁵-C₅H₅)Re(NO)(PPh₃)(CH₃) (1) with CF₃SO₃H and then NH₂CH₂C₆H₅ gives [(η⁵-C₅H₅)Re(NO)(PPh₃)(NH₂CH₂C₆H₅)]⁺ ([4a-H]⁺; 73%), and deprotonation with t-BuOK affords the amido complex (η⁵-C₅H₅)Re(NO)(PPh₃)(NHCH₂C₆H₅) (76%). Reactions of 1 with Ph₃C⁺ X⁻ and then primary or secondary amines give [(η⁵-C₅H₅)Re(NO)(PPh₃)(CH₂NHRR′)]⁺ X⁻ ([6-H]⁺ X⁻; R/R′/X = a, H/NH₂CH₂C₆H₅/BF₄; a′, H/NH₂CH₂C₆H₅/PF₆; b, H/NH₂CH₂(CH₂)₂CH₃/PF₆; c, H/(S)-NH₂CH(CH₃)C₆H₅/BF₄); d, CH₂CH₃/CH₂CH₃/PF₆; e, CH₂(CH₂)₂CH₃/CH₂(CH₂)₂CH₃/PF₆; f, CH₂C₆H₅/CH₂C₆H₅/PF₆; g, -CH₂(CH₂)₂CH₂-/PF₆; h, -CH₂(CH₂)₃CH₂-/PF₆; i, CH₃/CH₂CH₂OH/PF₆ (62-99%). Deprotonations with t-BuOK afford the amines (η⁵-C₅H₅)Re(NO)(PPh₃)(CH₂NRR′) (6a-i; 99-40%), which are more stable and isolated in analytically pure form when R ≠ H. Enantiopure 1 is used to prepare (R_ReS_C)-[6c-H]⁺, (R_ReS_C)-6c, (S)-[6g-H]⁺, and (S)-6g. The crystal structures of [4a-H]⁺, a previously prepared NH₂CH₂Si(CH₃)₃ analog, [6a′,d,f,h-H]⁺, (R_ReS_C)-6c, and 6f are determined and analyzed in detail, particularly with respect to cation/anion hydrogen bonding and conformation. In contrast to analogous rhenium containing phosphines, 6a-i show poor activities in reactions that are catalyzed by organic amines.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

X-ray diffraction studies on cation-collapsed DNA

The polyamines spermidine, spermine and putrescine are now known to induce tertiary collapse of DNA. In this collapsed state DNA assumes a compact toroidal conformation. However, the structural details of DNA in these compact particles and the forces that stabilize the collapsed state are not clear. We show here that the structural arrangement of DNA in this tertiary conformation is determined by the chemical structure of the agent used to collapse. We have used aliphatic triamines (NH₃⁺-(CH₂)₃-NH₂⁺-(CH₂)_n-NH₃⁺ with n = 3, 4, 5 and 8) and diamines (NH₃⁺-(CH₂)_x-NH₃⁺ with x = 2, 3, 4 and 6) to collapse DNA. We find that the Bragg spacing and the calculated interhelical spacing for a hexagonal packing model vary systematically with the length of the methylene bridge. We also find that the ionic strength of the solution has no effect on the Bragg spacing. This observation suggests that the arrangement of DNA strands in the complexes is determined by the structure of the polycation, and argues against suggestions that the structure of the collapsed state is maintained by the balance of long-range electrostatic repulsive and attractive forces. Instead we propose that DNA helices form a hexagonal array with counterions in the interstices between the helices resulting in a stable three-dimensional phase with high structural order. Arguments are presented favoring such a model in terms of stabilizing and destabilizing thermodynamic forces.     

Synthesis and antiproliferative activity of 2,7-diamino l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives as potent telomeric G-quadruplex DNA ligands

A novel series of l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives with terminal ammonium substituents at C2 and C7 positions on the acridone ring were successfully synthesized as antiproliferation agents. The biologic activity of the acridone compounds against leukemia CCRF-CEM cells demonstrated that some of the compounds displayed good antiproliferative activity, among which compound 6a containing dimethylamine substituents at the terminal C2 and C7 positions exhibited the highest cytotoxicity with IC₅₀ at 0.3 μM. In addition compound 6a showed little toxicity against normal 293T cells proliferation with IC₅₀ more than 100 μM. Further study indicated that compound 6a had strong binding activity to human telomeric G-quadruplex DNA, as detected by mass spectrometry, CD spectroscopy, UV absorption, FRET and fluorescence quenching assays. Our data suggested that the activity of 6a might be associated with its stabilization of G-quadruplex DNA, which can be developed as potent antitumor agent.     

Cytotoxicity of synthesized 1,4-naphthoquinone analogues on selected human cancer cell lines

In an effort to establish new candidates with enhanced anticancer activity of 5-hydroxy-7-methyl-1,4-naphthoquinone scaffold (7-methyljuglone) previously isolated from the root extract of Euclea natalensis, a series of 7-methyljuglone derivatives have been synthesized and assessed for cytotoxicity on selected human cancer lines. These compounds were screened in vitro for anticancer activity on MCF-7, HeLa, SNO and DU145 human cancer cell lines by MTT assay. Most of them exhibited significant toxicity on cancer cell lines with lower IC₅₀ values. The most potent derivative (19) exhibited the toxicity on HeLa and DU145 cell lines with IC₅₀ value of 5.3 and 6.8 μM followed by compound (5) with IC₅₀ value of 10.1 and 9.3 μM, respectively. Structure–activity relationship reveals that the fluoro substituents at position C-8 while hydroxyl substituents at C-2 and C-5 positions played an important role in toxicity.     

Specificity of the interaction of DNA-platinum, amount of platinum, and pH measurement]

Interaction between the sodium salt of a DNA extracted from salmon sperm (41% GC) with [Pt(NH₃)₄]Cl₂, [Pt(NH₂? (CH₂)₂? NH? (CH₂)₂? NH₂Cl]Cl, cis-Pt(NH₂? (CH₂)₂? NH₂)Cl₂, cis-Pt(NH₃)₂Cl₂, trans-Pt(NH₃)₂Cl₂, K[Pt(C₂H₄)Cl₃], and K₂[PtCl₄) indicates at least three types of complexation. A correlation is found between the change of pH and the number of platinum atoms fixed per (AT + GC) unit. The first binding site is located on the G-C pairs (guanine–cytosine), most likely the N-7(G) site, as it was shown in a previous study of the guanosine-platinum salts. The fixation of the second platinum atom by the pair (AT + GC) takes place with liberation of protons. In the case of the complexes cis-Pt(NH₂? (CH₂)₂? NH₂)Cl₂, cis-Pt(NH₃)₂Cl₂, and trans-Pt(NH₃)₂Cl₂ the second interaction seems to involve simultaneously the N-7(A) and the N-1(G) and N-3(C) sites. This latter intercrosslink between guanine and cytosine obviously liberates protons and the decrease of pH is related in this case to the trans effect of the platinum compounds. The first two platinum atoms in the reaction of K₂PtCl₄] or the Zeise salt, K[Pt(C₂H₄)Cl₃] with DNA are fixed on the G-C pairs. A maximum of six platinum atoms per (AT + GC) unit were fixed in this case. Preliminary experiments with a DNA extracted from bacteria Micrococcus lysodeikticus (72% GC) give similar results.     

Synthesis of metal complexes of peptide-phosphinites: Metal complexes in asymmetric Heck reaction

The synthesis of seven peptide-derived phosphinites, N-Boc-Phe-Tyr(OPPh₂)-OMe (4), N-Boc-Phe-Tyr(OPEt₂)-OMe (5), N-Boc-Phe-Tyr(OPCy₂)-OMe (6), N-Boc-Phe-Ser(OPPh₂)-OMe (7), N-Boc-Phe-Ser(OP^tBu₂)-OMe (8), N-Boc-Phe-Thr(OPPh₂)-OMe (9), N-Boc-Phe-Thr(OP^tBu₂)-OMe (10) is reported. These ligands are readily coordinated to Pd(II) and Pt(II) centers giving the corresponding complexes of the type ML₂Cl₂ (11-20). The palladium complexes, [N-Boc-Phe-Tyr(OPPh₂)-OMe]₂PdCl₂ (16), [N-Boc-Phe-Tyr(OPEt₂)-OMe]₂PdCl₂ (17), [N-Boc-Phe-Tyr(OPCy₂)-OMe]₂PdCl₂ (18), [N-Boc-Phe-Ser(OPPh₂)-OMe]₂PdCl₂ (19) and [N-Boc-Phe-Thr(OPPh₂)-OMe]₂PdCl₂ (20) catalyze the asymmetric phenylation of 2,3-dihydrofuran in moderate to high yields with high ee’s. The steric and electronic influences of the ligand substituents in driving the catalytic process are also discussed.     

Compacting effect of BBR3464, a new-generation trisplatinum anticancer agent,on DNA

BBR3464 is a trinuclear platinum compound of formula [{trans-PtCl(NH₃)₂}₂-μ-trans-Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂]⁴⁺. It is a new-generation platinum chemotherapeutic agent that exhibits cytotoxicity at ten to thousand times lower dose limit compared to the well-known platinum drug cisplatin, in cisplatin-sensitive as well as in cisplatin-resistant cells. DNA is thought to be the primary cellular target of BBR3464. In this work, we have applied high-resolution atomic force microscopy (AFM) for the first time, to obtain direct information on BBR3464-induced structural changes of DNA. It is found that the DNA molecules get compacted after treatment with BBR3464, for the drug:DNA molar ratio and the drug treatment period of 0.01 and 48 h, respectively. These values of molar ratio and incubation period have been obtained previously, as a result of biochemical optimization studies carried out for achieving maximum drug effects. The DNA structural changes, as observed in AFM topographs, have been correlated to the bulk level spectroscopic information. A remark on the significance of BBR3464-induced DNA compaction with respect to the available AFM reports on DNA modification by cisplatin has been made.     

The scope of thallium nitrate oxidative cyclization of chalcones; synthesis and evaluation of isoflavone and aurone analogs for their inhibitory activity against interleukin-5

The oxidative cyclization of 2′-hydroxy-6′-cyclohexylmethoxychalcones 5 using thallium (III) nitrate (TTN) in alcoholic solvents produced isoflavones 2 and (or) aurones 3 depending on the electronic nature of p-substituents on ring B. Chalcones with strong electron donating substituents (OH, OCH₃) were exclusively converted to isoflavones 2. Chalcone with weak electron donating substituents (CH₂CH₃) was transformed into isoflavone 2 and the aurone 3 in approximate ratio 1:1. Chalcones with hydrogen or electron withdrawing substituents (Cl, CHO, COOCH₃, and NO₂) formed aurones 3. Synthesized isoflavones 2 and aurones 3 were evaluated for their inhibitory activity against interleukin-5. Among them, 5-(cyclohexylmethoxy)-3-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (2h, >100% inhibition at 50 μM, IC₅₀ = 6.1 μM) gave most potent activity. All the aurones 3 were inactive.     

Effects of geometric isomerism in dinuclear antitumor platinum complexes on their interactions with N-acetyl-L-methionine

In this study, the reactions of N-acetyl-L-methionine (AcMet) with [{trans-PtCl(NH₃)₂}₂-μ-H₂N(CH₂)₆NH₂](NO₃)₂ (BBR3005: 1,1/t,t 1) and its cis analog [{cis-PtCl(NH₃)₂}₂-μ-{H₂N(CH₂)₆NH₂}]Cl₂ (1,1/c,c 2) were analyzed to determine the rate and reaction profile of chloride substitution by methionine sulfur. The reactions were studied in PBS buffer at 37°C by a combination of multinuclear (¹⁹⁵Pt, {¹H-¹⁵N} HSQC) magnetic resonance (NMR) spectroscopy and electrospray ionization time of flight mass spectrometry (ESITOFMS). The diamine linker of the 1,1/t,t trans complex was released as a result of the trans influence of the coordinated sulfur atom, producing trans-[PtCl(AcMet)(NH₃)₂]⁺ (III) and trans-[Pt(AcMet)₂(NH₃)₂]²⁺ (IV). In contrast the cis geometry of the dinuclear compound maintained the diamine bridge intact and a number of novel dinuclear platinum compounds obtained by stepwise substitution of sulfur on both platinum centers were identified. These include (charges omitted for clarity): [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{cis-Pt(AcMet)(NH₃)₂}] (V); [{cis-Pt(AcMet)(NH₃)₂}₂-μ-NH₂(CH₂)₆NH₂] (VI); [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)NH₃] (VII); [{PtCl(AcMet)(NH₃)}₂-μ-NH₂(CH₂)₆NH₂] (VIII); [{trans-Pt(AcMet)₂(NH₃)}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)(NH₃)] (IX) and the fully substituted [{trans-Pt(AcMet)₂(NH₃)}₂-μ-{NH₂(CH₂)₆NH₂] (X). For both compounds the reactions with methionine were slower than those with glutathione (Inorg Chem 2003, 42:5498–5506). Further, the 1,1/c,c geometry resulted in slower reaction than the trans isomer, because of steric hindrance of the bridge, as observed previously in reactions with DNA and model nucleotides.     

Antitumor bifunctional dinuclear PtII complex BBR3535 forms interduplex DNA cross-links under molecular crowding conditions

When antitumor platinum drugs react with DNA they form various types of intrastrand and interstrand cross-links (CLs). One class of new antitumor platinum compounds comprises bifunctional Pt^II compounds based on the dinuclear or trinuclear geometry of leaving ligands. It has been shown that the DNA-binding modes of dinuclear or trinuclear bifunctional Pt^II agents are distinct from those of mononuclear cisplatin, forming markedly more intramolecular interstrand CLs. However, at least two types of DNA interstrand cross-linking by bifunctional Pt^II complexes can be envisaged, depending on whether the platinum complex coordinates to the bases in one DNA molecule (intramolecular interstrand CLs) or in two different DNA duplexes (interduplex CLs). We hypothesized that at least some antitumor bifunctional poly(di/tri)nuclear complexes could fulfill the requirements placed on interduplex DNA cross-linkers. To test this hypothesis we studied the interduplex cross-linking capability of a representative of antitumor polynuclear agents, namely, dinuclear Pt^II complex [{trans-PtCl(NH₃)₂}₂-μ-{trans-(H₂N(CH₂)₆NH₂(CH₂)₂NH₂(CH₂)₆NH₂)}]⁴⁺ (BBR3535). The investigations were conducted under molecular crowding conditions mimicking environmental conditions in the cellular nucleus, namely, in medium containing ethanol, which is a commonly used crowding agent. We found with the aid of native agarose gel electrophoresis that the DNA interduplex cross-linking efficiency of BBR3535 under molecular crowding conditions was remarkable: the frequency of these CLs was 54%. In contrast, the interduplex cross-linking efficiency of mononuclear cisplatin or transplatin was markedly lower (approximately 40-fold or 18-fold, respectively). We suggest that the production of interduplex CLs in addition to other DNA intramolecular adducts may provide polynuclear Pt^II compounds with a wider spectrum of cytotoxicity.     

A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs

A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2′-deoxy, 2′-NH₂ and 2′-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2′-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2′-substituents (2′-OMe and 2′-N₃), and may also enhance use of NTPs with modifications at other than the 2′-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.     

Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time,Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis

Background

To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC₅₀ values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.

Results

Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC₅₀ and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC₅₀ and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05).

Conclusion

Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.     

Synthesis and anticancer activity of dichloroplatinum(II) complexes of podophyllotoxin

A series of dichloroplatinum(II) complexes of podophyllotoxin (PPT) were prepared, and their cytotoxicity against sensitive (A-549, HeLa, HCT-8, Hep-G2, K562) and resistant (ADM/K562) cell lines were evaluated. Complex cis-[4α-O-(2″,3″-diaminopropanoyl)-podophyllotoxin] dichloride platinum(II) (12) displayed most potent cytotoxicity with IC₅₀ value in the range 0.071–2.98 μM. Complex 12 induces cell cycle arrest in the G₂/M phase, and inhibits the formation of microtubules in HeLa cells. Furthermore, this complex exhibits potent DNA cleavage capabilities.