Patent Grant
8884000
The present invention relates to inhibitors of saporin-L1 and related compositions and uses thereof, in particular in cancer therapy that employs saporin-L1-linked immunotoxins.
Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Ribosome inactivating proteins (RIPs) are N-glycohydrolases that catalyze the depurination of adenosine A4234 from the highly conserved sarcin-ricin loop of the 28S eukaryotic ribosomal subunit RNA (1). Depurination inhibits the binding of elongation factor 2 to the ribosome, halts protein synthesis, and causes cell death (2). RIPs with broad polynucleotide:adenosine glycosidase activity can target other ribosomal sites and non-ribosomal substrates, including DNA, RNA and poly(A) (3, 4). Saporin-L1 toxin, a RIP from the leaves of the Saponaria officinalis plant can release adenine from poly(A), herring sperm DNA, tRNA, Escherichia coli rRNA, and globin mRNA at physiologic pH (5, 6). Fifteen saporin isoforms have been characterized from Saponaria officinalis including 9 seed, 3 leaf, and 3 root RIPs. These isoforms differ in ribosome translation inhibition activities and nascent cellular toxicity (7). Analysis of 50 type I and II RIPs revealed only saporin-L1 with the ability to release adenine from RNA of MS2, TMV, and AMCV viruses at physiologic pH, a catalytic activity unique in the RIP family of enzymes (3).
Transition state structures have been solved for the ricin A-chain, a potent RIP from castor beans (8, 9, 10). Kinetic isotope effect studies established that ricin A-chain hydrolysis of 10-mer RNA and DNA stem-loop substrates occurs through leaving group activation and forms ribooxacarbenium ion transition states (9, 10). Small RNA stem-loops are substrates for RIPs, and stem-loop substrate mimics of the sarcin-ricin loop serve as inhibitor scaffolds. Substrate stem-loops contain a GAGA tetraloop for RIP recognition and alternating C-G base pairs for stem stability and loop folding. Transition state analogues for ricin A-chain featured protonated 1-aza sugars to mimic the oxacarbenium ion intermediate and leaving groups with an elevated pKa at the depurination site (8). Ricin A-chain shows robust catalytic activity on stem-loop RNA substrates at pH 4 but is inactive with these substrates at neutral pH. Transition state analogues of ricin A-chain are nanomolar inhibitors at pH 4 but do not protect ribosomes from ricin A-chain action at neutral pH.
Ribosome inactivating proteins have potential as anticancer agents when linked to an appropriate recognition motif (11). Powerful inhibitors of the RIP can then provide a rescue agent to prevent the post-therapy vascular leak syndrome commonly associated with RIP immunochemotherapy (12). Such inhibitors could provide extracellular protection against circulating toxins. The present invention addresses the need for providing inhibitors of ribosome inactivating proteins that function at physiological pH and that can be used in cancer therapy that employs saporin-L1-linked immunotoxins.
The present invention provides small, stable and tight-binding transition state inhibitors of saporin-L1 that are effective at physiological pH, and pharmaceutical compositions comprising the inhibitors and a pharmaceutically acceptable carrier.
The invention also provides a method for inhibiting the activity of saporin-L1 comprising contacting saporin-L1 with any of the saporin-L1 inhibitors disclosed herein.
The invention further provides a method for treating or preventing a side effect in a subject undergoing chemotherapy with saporin-L1 attached to a targeting agent, the method comprising administering to the subject a therapeutically effective amount of any of the inhibitors of saporin-L1 disclosed herein.
The invention provides a transition state inhibitor of saporin-L1 selected from the group consisting of:
wherein each R is independently OCH3 or aryl or alkyl, where the aryl or alkyl can be optionally substituted with one or more of OH, ═O, OCH3, NH2, C1-C6 alkyl or halogen;
or a pharmaceutically acceptable salt of any of inhibitors i)-viii).
The inhibitor comprising the structure:
can comprise, for example, additional X-X′ pairs wherein in each pair, X and X′ are independently C-G, G-C, A-T, T-A, U-A, or A-U, or an analogue thereof, or any base-paired nucleotides or their analogues that form a stable stem-loop nucleic acid structure; wherein A, C and G are independently DNA, RNA, or RNA containing a 2′-OCH3 substitution; and wherein U can contain a 2′-OCH3 substitution. The inhibitor can comprise, for example, X4-X4′, or X4-X4′ and X5-X5′. The inhibitor can consist or consist essentially of the structure.
The inhibitor can, for example, comprise or have the structure:
“A” represent the nucleotide adenylate (in RNA) or deoxyadenylate (in DNA). “C” represents the nucleotide cytidylate (in RNA) or deoxycytidylate (in DNA). “G” represents the nucleotide guanylate (in RNA) or deoxyguanylate (in DNA). “T” represent the nucleotide thymidylate in DNA. “U” represents the nucleotide uridylate in RNA. In one embodiment, A, C and G are DNA. In another embodiment, A, C and G are RNA. In DNA, hydrogen (H) is attached to the 2′ carbon of the pentose (i.e., 2-deoxyribose). In RNA, H and —OH are attached to the 2′ carbon of the pentose (i.e., ribose). In one embodiment of the invention, at least one A, C or G is RNA and contains a —OCH3 (i.e., —OMe or —OMethyl) substitution at the 2′ carbon of the pentose ring.
In inhibitor structure ii), R is OCH3 or an aryl or alkyl protecting group to prevent the action of ribonuclease degradation of the inhibitor. Preferably, the alkyl is C1-C6 alkyl, and more preferably, C1-C3 alkyl. Preferably, the aryl is phenyl or a phenyl substituted with one or more of —CH3, —OH, —OCH3, —F, —Cl, —Br, or —I.
Preferred inhibitors include those illustrated in
The present invention also provides a pharmaceutically acceptable salt of any of the inhibitors disclosed herein. Pharmaceutically acceptable salts include non-toxic salts derived from inorganic or organic acids, such as, for example, the following salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.
The invention also provides a pharmaceutical composition comprising any of the inhibitors disclosed herein and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable” carrier means a material that (i) is compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, sterile isotonic saline, water, and emulsions such as, for example, oil/water emulsions and microemulsions.
The invention also provides a method for inhibiting the activity of saporin-L1 comprising contacting saporin-L1 with any of the inhibitors disclosed herein. The present inhibitors are active at physiological pH. Accordingly, the method can be carried out at a pH of 7.3-7.5. The method can be carried out, for example, in a subject who has been poisoned with saporin-L1 toxin.
The invention further provides a method for treating or preventing a side effect in a subject undergoing chemotherapy with saporin-L1 linked or attached to a targeting agent, the method comprising administering to the subject a therapeutically effective amount of any of the inhibitors of saporin-L1 disclosed herein. Saproin-L1 can be linked or attached to a targeting agent such as an antibody, antibody fragment (e.g., F(ab′)2 or Fab′ fragment), peptide or nucleic acid aptamer, and used to treat a disease such as cancer. The side effect can be associated with vascular leak syndrome (VLS). VLS can occur following introduction of saproin-L1 into the circulation, which could occur for example following tumor lysis. Increased vascular permeability in VLS results in extravasation of fluids and proteins. The side effect can be one or more of fluid retention, increase in body weight, hypotension, peripheral edema, ascites, hypoalbuminemia, oliguria, dyspnea, respiratory insufficiency, pleural or pericardial effusion, or in severe cases pulmonary or cardiovascular failure. Administration of the inhibitors of saporin-L1 described herein can be used to prevent or treat vascular leak syndrome in a subject undergoing chemotherapy with a saporin-L1-linked immunotoxin for treatment of cancer. As used herein, to “treat” a side effect means to minimize or reverse the side effect.
The inhibitors of the present invention can be administered to subjects using routes of administration known in the art. The administration can be systemic or localized to a tumor site. Routes of administration include, but are not limited to, intravenous, intramuscular, intrathecal or subcutaneous injection, oral or rectal administration, and injection into a tumor site. Intravascular administration is a preferred route of administration.
The subject is preferably a mammal and most preferably a human.
This invention will be better understood from the Experimental Details that follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Introduction
Saporin-L1 has not been kinetically characterized with small substrates or inhibitors. Here its kinetic properties are characterized on small stem-loop substrates and on mammalian ribosomes, and novel transition state analogue inhibitors are provided, all which function at physiological pH values. Kinetic analysis takes advantage of a sensitive and continuous assay for adenine linked to luciferase-based light production (13). Saporin-L1 catalyzes the depurination of adenines from A-10 (an RNA stem-loop 5′-CGCGAGAGCG-3′ (SEQ ID NO:1) mimic of the sarcin-ricin loop), linear and covalently closed circular constructs related to A-10, and mammalian 80s ribosomes, all at physiologic pH. The transition state (TS) mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA) replacement for adenosine in the RIP recognition GAGA tetraloop motif inhibits saporin-L1 catalysis. Inhibitors were synthesized against saporin-L1, including monomer to 14-mer oligonucleotides, employing the DADMeA transition state mimic. Stem-loop inhibitor constructs, cyclic tetramer 5′- to 3′-covalently closed circular GAGA tetraloops, and monomeric inhibitors were also applied to saporin-L1. A minimal saporin-L1 inhibitor scaffold with 2′-OMe containing compounds designed to give enhanced nuclease stability provided inhibition of saporin-L1 to low nanomolar Ki values and also protected ribosomes from saporin-L1 action in rabbit reticulocyte lysates.
Materials and Methods
Materials. Oligonucleotide A-10 was purchased from Dharmacon (Lafayette, Colo.). 5′-DMT protected 9-DA azasugar was synthesized and purified as previously described (8). DNA/RNA synthesis reagents were purchased from Glen Research (Sterling, Va.) and ChemGene Co. (Ashland, Mass.). HPLC purifications were preformed on a Waters 626 pump with a 996 photodiode array detector with Millennium software. Firefly luciferase ATP assay kit (ATPlite) was purchased from Perkin Elmer (Waltham, Mass.). Phosphatase inhibitors (PhosSTOP) were purchased from Roche Applied Science (Indianapolis, Ind.). RNase inhibitor (SuperRNasin) was purchased from Ambion (Austin, Tex.). Ricin A-chain and Saporin-S6 was purchased from Aldrich Chemical Corp. (Ashland, Mass.). For translation assays, FlexiRabbit Reticulocyte Lysate System, luciferase assay system, and rabbit reticulocyte lysate (untreated) were purchased from Promega (Madison, Wis.). Buffers and enzyme preparations were checked for RNase activity using RNaseAlert from Ambion (Austin, Tex.). DEPC treated water (0.1% DEPC stirred for 20 min followed by 30 min autoclave treatment) was used in all enzymatic reactions and buffers. All other reagents used were purchased in the highest purity available from Fisher Scientific (Pittsburgh, Pa.) or Aldrich Chemical Corp. (Ashland, Mass.). Concentrations of adenine and oligonucleotides were measured using a Nanoprop 1000 (Thermo Fisher Scientific, Waltham, Mass.). Inhibitor concentrations were determined spectrophotometrically including the published millimolar extinction coefficient of 8.5 at 275 nm at pH 7 for 9-deazaadenosine (14). Enzyme concentrations of saporin-L1 and saporin-S6 were determined with the BCA protein assay kit from Pierce (Rockford, Ill.). Luminescence measurements were accomplished on a GloMax 96-well luminometer from Promega (Madison, Wis.).
Saporin-L1 Isolation. Saporin-L1 was isolated from the leaves of Saponaria officinalis (common soapwort) as described previously with modifications described below (7). Freshly harvested leaves (10 g) were frozen and ground with a pestle under liquid nitrogen. The powder was suspended in 80 mL extraction buffer (10 mM Na2HPO4 (pH 5.5) (titrated with citric acid), 175 mM NaCl, 2.5 mM MgCl2, 1 mM CaCl2, one tablet of complete protease inhibitor (Roche), 1% (w/v) poly(vinylpolypyrrolidone), 1% cellulase, 0.5% hemicellulase, and 150 units pectinase). The mixture was stirred at room temperature for 3 hours and then acidified to pH 4.0 with acetic acid. Triton X-100 was added to 0.5% (v/v) and the mixture was stirred for an additional 1 hour. The digested and lysed leaf mixture was then filtered through cheese cloth and centrifuged at 25,000 g for 30 minutes. The supernatant was loaded on SP-sepharose FF resin (Amersham) pre-equilibrated in 10 mM sodium phosphate, pH 4.5. The column was extensively washed with 10 mM sodium phosphate pH 7.4 and the crude saporin-L1 containing fraction was eluted with the same buffer containing 1 M NaCl. The elute was dialyzed against 10 mM sodium phosphate pH 7.4 titrated to pH 4.5 with acetic acid and loaded onto carboxymethyl-FF (three 1 mL columns, Amersham) pre-equilibrated in buffer [10 mM sodium phosphate pH 7.4]. The columns were washed extensively with buffer to achieve pH equilibration. Saporin-L1 was eluted with a 50 min linear gradient of 0-300 mM NaCl in 10 mM sodium phosphate pH 7.4 at 1 ml/min and was identified as an ˜30 kD band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Saporin-L1 fractions were combined, titrated to pH 4.5 with acetic acid, and loaded onto heparin HP (three 1 mL columns, Amersham) pre-equilibrated in buffer. Saporin-L1 was eluted with a 50 minute linear gradient of 0-800 mM NaCl in 10 mM sodium phosphate pH 7.4 (1 ml/min). Saporin-L1 was eluted as the last major peak in the chromatogram and was identified by SDS-PAGE. The >80% pure Saporin-L1 was concentrated by spin Amicon concentrator and purified to >95% homogeneity with a BioSep-SEC-S 2000 column (Phenomenex) equilibrated in 20 mM sodium phosphate pH 7.4 and eluted at 1 mL/min. The gel filtration purification step was needed to remove trace DNAase and RNAase activities from Saporin-L1. Saporin-L1 was concentrated to ˜1 mg/mL and stored at 4° C. The yield was ˜0.5 mg saporin-L1 from 10 g of leaf material. Commercial saporin-S6 was purified with the heparin chromatography step as described above.
Saporin-L1 Seed Isolation. 50 g of Saponaria officinalis (common soapwort) seeds were frozen in liquid nitrogen and ground to a powder in a seed grinder. The powder was added to 350 mL of PBS and stirred overnight at 4° C. The mixture was filtered through cheese cloth, centrifuged at 25,000 g for 30 minutes, and extensively dialyzed against 10 mM Na2HPO4/NaH2PO4 pH 7.0. The cloudy solution was acidified to pH 4.5 with acetic acid, and clarified with centrifugation at 5000 g for 10 minutes. The supernatant was collected and loaded onto a heparin column (20 mL Amersham) and crude SAP-L1 was eluted in the end fractions with a gradient of 0-500 mM NaCl in 10 mM Na2HPO4/NaH2PO4 pH 7.4 over 17 columns. Because SAP-L1 was a minor protein within the seed preparations with similar migration on SDS-Page to seed isoforms, the fastest catalyzing fractions of 50 μM A-10 RNA substrate (containing SAP-L1) were also identified by an activity based assay using the adenine lucifease assay for adenine quantification. Crude SAP-L1 was concentrated in an Amicon, dialyzed against 10 mM Na2HPO4/NaH2PO4 pH 7.4 and further purified as described for leaf derived protein. Final isolation yields were 1 mg of saporin-L1 per 50 grams of seed.
Saporin-L1 N-terminal Sequencing and Mass Analysis. Purified saporin-L1 from the leaves of Saponaria officinalis was verified by N-terminal sequencing at the Rockefeller University Proteomics Resource Center (New York, N. Y.). The N-terminal sequence was VIIYELNLQG (SEQ ID NO:2), which matched previous reports for the Saporin-L1 isoform (7). The mass of saporin-L1 was measured on a MALDI-TOF mass spectrometer in the linear positive ion mode with external calibration. Protein samples (˜20 μM) were desalted with a ZipTip (Waters) as described by the manufacturer and eluted onto a 100 well gold plate with 1 μL of matrix solution (20 mg/mL sinapic acid in 70% acetonitrile/H2O with 0.1% TFA). The mass of saporin-L1 (28,749 Da) isolated from the leaves of Saponaria officinalis was comparable to previous reported masses for saporin-L1 leaf and vacuolar isoforms (28,740 to 28,765 Da) (15).
Synthesis of Oligonucleotides Inhibitors. Cyclic oligonucleotides and 1-aza sugar phosphoramidites were synthesized and purified as reported previously (16). Stem-loop oligonucleotides were synthesized on a 1 μmol scale with DMT-on mode using an Expedite 8909 DNA/RNA synthesizer following standard synthesis protocols for β-cyanoethyl phosphoramidite chemistry with acetyl protected cytosine phosphoramidite and 5-benzylthio-1H-tetrazole as activator. Cleavage from solid support and base deprotection of a 1 μmol synthesis was accomplished in 1.5 mL AMA reagent (1:1 concentrated NH4OH to 40% aqueous methylamine) for 45 minutes at 37° C. The reaction mixture was centrifuged, the supernatant collected, and the resin was washed twice with 3:1:1 ethanol:acetonitrile:water. Combined supernatant and washes were evaporated to dryness under vacuum. 2′-O-TBDMS deprotection of the A-14 (DADMeA) RNA (1 μmole) was accomplished using 250 μL of anhydrous TEA HF/NMP solution (1.5 mL of N-methylpyrrolidinone, 750 μL of TEA, and 1.0 mL of TEA-3HF) heated to 65° C. for 2 h (17). This reaction mixture was diluted with 2 mL of 0.5 M NH4OAc and evaporated to dryness under vacuum.
HPLC purification of the 5′-trityl stem-loop oligonucleotides was accomplished to >95% purity on a Waters Delta-Pak (7.9 mm×300 mm) semipreparative C18 reversed phase column at 3.5 mL/min in 20 mM NH4OAc/5% CH3CN with a linear 0-40% gradient of CH3CN in 25 min. Trityl protected oligonucleotides were the major peak and eluted at ˜25 min. The major late eluting fraction was evaporated to dryness under vacuum. The pellet was dissolved in 1 mL of 80% acetic acid in water, incubated at 30° C. for 1 hour, and the solution was evaporated to dryness under vacuum. HPLC purification of the final oligonucleotide was accomplished to >95% purity on a Waters Delta-Pak (7.9 mm×300 mm) semipreparative C18 reversed phase column at 3.5 mL/min in 50 mM triethyl ammonium acetate pH 7.0 with a linear 0-80% gradient of 50% aqueous methanol in 40 min. The final product was evaporated to dryness in a speed vac concentrator and resuspended in sterile RNAase free water.
Linear inhibitors were synthesized in DMT-off mode on an Expedite 8909 synthesizer in otherwise identical conditions to stem-loop oligonucleotides. After deprotection in AMA, the oligonucleotides were purified by HPLC to >95% purity as described for stem-loops in 50 mM triethyl ammonium acetate pH 7.0 with a linear 0-50% gradient of 50% aqueous methanol in 40 to 60 min.
Stem-loop, cyclic, and linear oligonucleotide structures were confirmed using a MALDI-TOF mass spectrometer as described previously (16). Observed and calculated masses for the final compounds are indicated in Table 1. Prior to use in inhibition assays, stem-loop oligonucleotides were heated to 95° C. for 1 minute and cooled on ice.
Saporin-L1 Kinetic Assay. Saporin-L1 kinetics on substrate A-10 RNA (5′-CGCGAGAGCG-3′) (SEQ ID NO:1) were determined using a continuous coupled assay for quantifying free adenine by linking it to the production of light from luciferase (13). In brief, an adenine detection buffer was prepared in bulk (50 mL of charcoal-filtered solution containing 100 mM tris-acetate pH 7.7, 2 mM phosphoenolpyruvic acid, 2 mM sodium pyrophosphate, 2 mM 5-phospho-D-ribosyl-1-pyrophosphate (PRPP), 15 mM NH4SO4, 15 mM (NH4)2MoO4, and phosphatase inhibitors in RNAase free water) and stored at −80° C. in 1 mL aliquots. Prior to use in adenine assays, coupling enzymes were prepared by adding 10 mM MgSO4, 8 units of APRTase, 8 units of phosphoenolpyruvate dikinase, 200 μL D-luciferin/luciferase (ATPLite) reagent and 1 μL of SuperRNasin (Ambion) per 1 mL of coupling enzymes buffer. One unit of enzyme activity was defined as the amount that forms one mole of product per min at 20° C.
Varying concentrations of A-10 RNA (5′-CGCGAGAGCG-3′) (SEQ ID NO:1) were prepared in 1:1 diluted coupling enzymes in a 96-well luminometer plate and reactions were initiated with 300 μM saporin-L1 (50 μL total reaction). Luminescence was measured with a luminometer in kinetic acquisition mode for several minutes. Adenine standards were prepared in identical assay conditions. The initial rates of adenine formation were calculated by converting luminescent rate (lumens/second) to enzymatic rate (pmol adenine/min/pmol enzyme) calibrated from the adenine standard curve. Kinetic parameters kcat and Km were calculated by fitting initial rates to the Michaelis-Menten equation.
Rabbit ribosomes (80S) were purified from rabbit reticulocyte lysate by sucrose cushion centrifugation (13). Saporin-L1 (300 μM) was analyzed for kinetic parameters with ribosomes as substrate as described for A-10 RNA substrate. Ribosome concentration was determined by depurinating (to completion) two stock concentrations with 500 nM RTA and comparing the final luminescence to the adenine standard curve fit. RTA releases 1 mol adenine from 1 mol ribosome and thus provides a method of quantitation.
Saporin-L1 Inhibition Assays. Saporin-L1 inhibition constants for stem-loop, circular, and linear oligonucleotides were determined in a competition assay using RNA A-10 substrate with quantitative analysis of adenine release as described for kinetic assays. Varying concentrations of inhibitor were pre-incubated with 300 μM saporin-L1 for 10 min in 1× continuous assay buffer at 20° C. Reactions were initiated by the addition of A-10 (−80 μM) and light generation (RLU) was measured in a luminometer over several minutes to obtain the initial rates (lumens/sec). The maximum rate of catalysis (kcat) was calculated from the Michaelis equation as described in kinetic assays. In cases where slow-onset inhibition was observed, Ki* was used to define the inhibition. Pre-incubation of inhibitor with saporin-L1, followed by initiation of the reaction with substrate provided a direct measure of Ki*. Values for the inhibition constant (Ki*) were calculated by fitting post slow-onset rates to the equation for competitive inhibition, v=kcat[S]/[[S]+Km(1+I/Ki*)], where v is the initial reaction rate, [5] is the substrate concentration, Km is the Michaelis constant for A-10, and kcat is the initial rate at A-10 saturation. For tight inhibition, when the concentration of inhibitor was ≦5 times the enzyme concentration, a correction was made for free inhibitor concentration. The free inhibitor concentration was determined by the relationship I=It−(1−vi/vo)Et, where It is total inhibitor concentration, vi and vo are the inhibited and uninhibited steady-state rates, respectively, and Et is the total enzyme concentration.
Protein translation assays. Saporin-L1 inhibition of protein translation was determined using a reticulocyte lysate translation system to express luciferase from mRNA as described by the manufacturer. For IC50 determination, 30 μL translation reactions in triplicate with varying concentrations of saporin-L1 were incubated at 37° C. at 1.5 hr. A 10 μL aliquot was sampled and luminescence was measured with a luciferase detection kit (Promega) according to the manufacturer's protocol in a 96-well plate format on a luminometer. Percent translation relative to control was plotted versus the log of saporin-L1 concentration and fit to a dose-response curve for the calculation of IC50.
For EC50 determination, triplicate reactions of 2.1 nM saporin-L1 with increasing inhibitor concentrations were pre-incubated at room temperature for 10 min in 5 μL of buffer (20 mM tris-acetate pH 7.4, 25 mM KCl, 5 mM MgCl2). Translation mix (25 pt) was added (300 μM saporin-L1 final) to the pre-incubated samples and were incubated at 37° C. for 1.5 hours. A 10 μL aliquot was sampled and luminescence was measured with the luciferase detection kit (luminescence) as described above. A control with the maximum inhibitor concentration without saporin-L1 established that the oligonucleotide itself did not affect luciferase expression. Percent translation relative to control (no saporin-L1) was plotted versus the log of inhibitor concentration and was fit to a dose-response curve for the calculation of EC50.
Synthesis of saporin inhibitors based on JMI382. The compound JMI382(±) was converted to phosphoramidite, purified, and incorporated to three different oligonucleotide constructs (
These inhibitors were examined on Saporin L1 by continuous luciferase assay. Each inhibitor was assayed by using the diastereomer mix, without further investigation of the more active component. The Ki values are 43.2 nM, 67.9 nM, 27.9 nM respectively for the di-, tri- and tetra-nucleotides (
Experimental scheme and data for the SerMe building block used for the saporin inhibitors are indicated below.
(9H-Fluoren-9-yl)methyl (4-benzamido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropan-2-yl)carbamate 9 (JMI382):
N-(4-methoxybenzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine 2: Chloropyrimidine 1 (4.2 g, 27.3 mmol), 4-methoxybenzylamine (10.7 mL, 82 mmol) and ethanol (25 mL) were heated at 100° C. for 6 h. The still warm solution was diluted with methanol, stirred with charcoal and filtered through Celite. Addition of water and ammonia to pH9 gave a precipitate that was isolated by filtration and washed with water, methanol and dichloromethane to give the title amine 2 (3.8 g, 14.9 mmol, 54%) as a brown solid. 1H NMR (DMSO d-6) δ 10.91 (s, 1H), 8.18 (s, 1H), 7.48 (bs, 1H), 7.33 (m, 3H), 6.92 (m, 2H), 6.37 (m, 1H), 4.65 (m, 2H), 3.74 (s, 3H).
4-(4-Methoxybenzylamino)-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde 4: Pyrimidine amine 2 (3.8 g, 14.9 mmol), potassium carbonate (5.2 g, 37.4 mmol), aqueous formaldehyde (37%, 40 mL, 1.3 mol), dioxane (320 mL) and water (80 mL) were heated together at 90° C. for 40 min. The cooled solution was evaporated onto silica gel and hydroxymethylpyrimidine 5 (3.2 g, 11 mmol) was isolated by elution through a plug of silica gel with dichloromethane-methanolic ammonia (1.4N), (9:1 and 4:1) and precipitation from aqueous methanol. A portion (1.4 g, 4.9 mmol) of this material was dissolved in acetic acid (50 mL) and stirred with Dess-Martin periodinane (2.5 g, 5.9 mmol) for 1 h. The solution was concentrated to a small volume and poured into a mixture of sodium bicarbonate (10%, aqueous, 50 mL) and sodium thiosulphate (5%, aqueous, 50 mL). Filtration gave the title aldehyde 4 (1.5 g, 4.52 mmol, 69%) contaminated with 15 mol % of iodobenzoic acid salts. This material was used in the next step; a small sample was further purified by chromatography on silica eluted with dichloromethane-methanol (9:1). 1H NMR (DMSO d-6) δ 11.9 (bs, 1H), 10.11 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 7.62 (bs, 1H), 7.33 (m, 2H), 6.92 (m, 2H), 6.37 (m, 1H), 4.66 (m, 2H), 3.74 (s, 3H). 13C NMR (DMSO d-6) 184.6, 158.4, 152.3, 149.6, 144.7, 134.1, 130.7, 129.0, 116.7, 114.6, 113.9, 55.2, 42.9. ESI-MS+, found 283.1198, calc. for C15H15N4O2 [M+H]+ 283.1195.
2-((4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)propane-1,3-diol 6. Aldehyde 5 (1.5 g, 85%, 4.2 mmol) was suspended in methanol (100 mL) containing serinol (0.49 g, 5.4 mmol) and stirred at room temperature overnight. Sodium borohydride (170 mg, 4.5 mmol) was added to the resulting solution and stirring was continued for a further 30 min. The solution was evaporated to dryness. Chromatography of the residue on silica gel eluted with dichloromethane-methanolic ammonia (1.4 M) (1:1) gave serinol adduct 7 (0.9 g, 56%). This compound was dissolved in trifluoroacetic acid (12 mL) and heated in a microwave oven to 100° C. for 8 min. After cooling the solvents were removed under reduced pressure. Purification of the residue on a column of silica gel eluted with dichloromethane-methanolic ammonia (3.5 M), 2:1 and 1:1, gave the title compound as a pale yellow glassy solid (0.38 g, 1.6 mmol, 38%). 1H NMR (D2O) δ 7.88 (s, 1H), 7.24 (s, 1H), 3.76 (s, 2H), 3.62 (dd, J=5.5, 11.6 Hz, 2H), 3.54 (dd, J=5.6, 11.6, 2H), 2.77 (m, 1H). 13C NMR (D2O) 150.2, 149.6, 144.6, 128.6, 113.6, 112.0, 60.7, 58.9, 39.6. ESI-MS+, found 238.1307, calc. for C10H16N5O2 [M+H]+ 238.1304.
(9H-Fluoren-9-yl)methyl (4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl(1,3-dihydroxypropan-2-yl)carbamate 7: Amine 6 (330 mg, 1.4 mmol) was suspended in methanol (35 mL) and 10% aqueous NaHCO3 (3.5 mL). Fmoc chloride (432 mg, 1.7 mmol) was added and the solution stirred for 15 min. The suspension was concentrated under reduced pressure. Chromatography of the residue on a column of silica gel eluted with 7.5-20% methanol in dichloromethane gave the title compound 7 (150 mg, 0.33 mmol, 48% allowing for recovered starting material). Subsequent elution with 50% methanolic ammonia (3.5M) in dichloromethane returned the starting amine 6 (170 mg, 51%). 7, colourless amorphous solid, ESI-MS+, found 460.1975, calc. for C25H26N5O5 [M+H]+ 460.1985.
(9H-Fluoren-9-yl)methyl (4-benzamido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl(1,3-dihydroxypropan-2-yl)carbamate 8: Chlorotrimethylsilane (810 μL, 6.3 mmol) was added to a solution of 7 (194 mg, 0.42 mmol) in dry pyridine. After 30 min benzoyl chloride (172 μL, 1.48 mmol) was added and the solution was stirred overnight. The reaction was quenched by the addition of water and concentrated to dryness. The residue was taken up in dichloromethane (10 mL), washed with water, concentrated, stirred for 5 min in methanolic ammonia (7N) and concentrated again. Chromatography of the residue on silica gel eluted with 4% methanol in dichloromethane gave the title compound as a colourless, amorphous solid (160 mg, 2.84 mmol, 67%). ESI-MS+, found 564.2245, calc. for C32H30N5O5 [M+H]+ 564.2245.
(9H-Fluoren-9-yl)methyl (4-benzamido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropan-2-yl)carbamate 9 (JMI382): Compound 8 (220 mg, 0.39 mmol) was evaporated from pyridine and then redissolved in the same solvent (12 mL). Dimethoxytrityl chloride (160 mg, 0.47 mmol) was added and the solution was stirred for 48 h. Solvent was removed under reduced pressure and the residue partitioned between dichloromethane and NaHCO3 solution (10% aqueous). Evaporation of the solvent and chromatography of the residue on a column of silica gel eluted with ethyl acetate gave the title compound as an amorphous solid (170 mg, 0.20 mmol, 50.3%). ESI-MS+, found 866.3545, calc. for C53H48N5O7 [M+H]+ 866.3554. HPLC (Waters XBridge-C8, 60-100% CH3CN in water) RT 5.1 min, 98.4%.
Results and Discussion
Saporin-L1 Catalysis. Initial rate kinetics were measured by coupling the adenine product to a luciferase-luciferin coupled assay with quantitation via luminescence (13). Saporin-L1 catalyzed deadenylation of A-10 gave a hyperbolic saturation curve with a kcat=440±16 min−1 and Km=95±7 μM at pH 7.7 (Table 2). MALDI-TOF analysis of the saporin-L1 reaction product showed that both adenosines in the GAGA tetraloop of A-10 were depurinated during prolonged incubations. Cyclic oxime RNA GAGA, a circular oligonucleotide substrate, was also depurinated by saporin-L1 with kinetics comparable to A-10 RNA with a kcat=301±27 min−1 and Km=82±15 μM (Table 2). The synthetic linker in circular oxime GAGA substrates folds the tetraloop for RIP recognition and is proposed to mimic the structure of stem-loop oligonucleotides (
Saporin-S6 catalysis. A-10 catalysis by saporin-S6, an RIP from Saponaria officinalis seeds, was also measured. Saporin-S6 was a commercial preparation and was further purified to remove contaminating saporin-L1. Saporin-S6 and saporin-L1 co-eluted on carboxymethyl resin but were separated by heparin chromatography. Saporin-S6 initial rate catalysis of A-10 RNA as substrate gave a kat of 0.35±0.04 min−1 and Km of 360±60 μM at pH 7.7 (Table 2). Saporin-L1 catalyzes (kcat) A-10 RNA approximately 1,500 times faster and has a 4-fold lower Km than saporin-S6. Thus, saporin-L1 is 4.800-fold more efficient (kcat/Km) at catalyzing A-10 RNA depurination than saporin-S6 (Table 2). Ricin A-chain is incapable of a single turnover of A-10 above pH 6.5 but at pH 4.0 gives a kcat of ˜4 min−1 on this substrate (18). Saporin-S6 was previously reported to catalyze the depurination of a 35-mer synthetic SRL mimic with GAGA tetraloop at pH 7.6 with a kcat of 0.4 min−1 and Km of 9 μM, while RIPs trichosanthin, gelonin, cinnamomin A-chain, and ricin A-chain had no detectable activity at physiological pH values (19).
Action of Saporin-L1 on 80S Ribosomes. Saporin-L1 action on 80S rabbit reticulocyte ribosomes (40 nM) showed multiple adenines released with a rate of 50 min−1 which is 250-fold faster than adenine release from A-10 RNA at an equivalent concentration. A continuous, linear rate was observed for the formation of adenine from 80S rabbit ribosomes extending well past one adenine/ribosome. Thus, saporin-L1 lacks sarcin-ricin loop specificity as the primary ribosomal depurination target. Previous reports indicate that saporin-L1 depurinates up to 36 adenines/mol from 80S rat ribosomes while saporin-S6 releases 1 to 2.5 adenine/mol (20). Moreover, saporin-L1 was reported to release ˜6 adenines from 80S rat ribosome before 50% inhibition of protein synthesis was observed in in vitro translation assays with a poly(U) transcript (5). Thus, adenines other than those at the sarcin-ricin loop are removed preferentially. Most other ribosome inactivating proteins are highly specific in releasing one mole of adenine per mole ribosome from the eukaryotic sarcin-ricin loop (1). Thus, both ricin A-chain and saporin-S6 release only 1 mole adenine per mole 80S rabbit ribosome in the present assay conditions. Ribosomal proteins surrounding the sarcin-ricin loop are known to influence the ribotoxic mechanism of RIP substrate recognition (21, 22).
Saporin-L1 Inhibitors. DADMeA (9-DA in Figures) is a non-hydrolysable methylene bridged 9-deazaadenine 1-aza sugar with features of the transition state for depurination of A-10 constructs by ricin A-chain. The replacement of N for C at C1′ of the ribosyl group mimics the carbocation of the dissociated transition state (8, 10). Replacing adenine with 9-deazaadenine caused an elevated pKa at N7, another feature of the transition state. The methylene linker between the 9-deazaadenine and the hydroxypyrrolidine places the base analogue and ribocation at approximately the same distance as found at the transition state. The omission of the 2′-hydroxyl found in RNA is required for the chemical stability of 9-DA. Replacing the scissile adenosine with 9-DA within the 14-mer stem-loop [A-14 (9-DA) RNA] competitively inhibited saporin-L1 with A-10 RNA as substrate with a Ki* of 3.7±0.7 nM at pH 7.7 (Table 3). The slow onset inhibition (Ki*) observed for saporin-L1 binding of inhibitors such as A-14 (9-DA) RNA was common to all inhibitor constructs listed in Table 2 excluding s3(9-DA)s3 and 9-DA (
ASee FIG. 1 for structures of inhibitors.
BValues are Ki with no slow onset observed.
Circular Inhibitors. Cyclic DNA and cyclic 2′-OMe modified G(9-DA)GA oligonucleotides inhibited saporin-L1 with Ki* values of 2.3±0.1 nM and 3.9±0.5 nM respectively (Table 3,
Linear Inhibitors. Monomer, linear dimer, trimer and tetramer inhibitor scaffolds containing 9-DA in the RTA-scissile adenosine position were also investigated (
Saporin-L1 Translation Inhibition. Saporin-L1 inhibited the translation of luciferase mRNA by rabbit reticulocyte lysate in a cell-free translation assay with an IC50 of 45 pM (
Saporin-L1 Inhibitor Implications for Toxin Immunotherapy. Saporin-L1 immunotoxin conjugates have been shown to be equally cytotoxic to cells as saporin-S6 conjugates (27). The catalytic A-chains of saporin-S6 and ricin have been investigated as anticancer agents in phase I/II clinical trials using chimeric immunotoxin conjugates in the treatment of lymphomas (11, 28, 29, 30). Therapeutic uses of immunotoxins have been limited by the vascular leak syndrome, a side effect resulting from inappropriate toxin targeting to the capillary bed of endothelial cells, causing edema and multi-organ failure (12, 31, 32, 33). Thus, cancer therapies that employ RIP-linked immunotoxins remain a challenge. Inhibitors to RIPs could provide a way to rescue normal cells following treatment of a targeted tissue with immunotoxins.
Saporin-L1 translation inhibition rescue by TS inhibitor. Protein translation, its inhibition by saporin-L1, and its rescue by inhibitors were studied in rabbit reticulocyte preparations. The minimal saporin-L1 inhibitor dinucleotide s3(9-DA)Gs3 2′-OMe was used (
Saporin-L1 is highly active on mammalian ribosomes and potently inhibited by the transition state mimic DADMeA (9-DA) when incorporated into the depurination site of stem-loop, circular and linear nucleic acid scaffolds appropriate for RIP recognition. This activity occurs at physiological conditions. The oligonucleotide context is essential as 9-DA alone is a poor inhibitor. Small oligonucleotide inhibitors featuring DADMeA bind up to 40,000 fold tighter than small stem-loop RNA substrates. Nine inhibitors exhibited slow onset binding to saporin-L1 with dissociation constants from 8.7 to 2.3 nM. Inhibitors constructed on RNA, 2′-OMe, and DNA scaffolds supported 9-DA binding with only small scaffold effects. Covalently closed circular inhibitors constructed on DNA or 2′-OMe RNA scaffolds were also excellent inhibitors. Dinucleotide s3(9-DA)Gs3 was the smallest tight binding inhibitor of saporin-L1. In ribosome translation rescue assays, s3(9-DA)Gs3 restored protein synthesis by inhibiting saporin-L1 depurination activities. Direct competition assays between 80S rabbit ribosomes and s3(9-DA)Gs3 established saporin-L1 inhibition with a dissociation constant of 7.8 nM.
Transition state inhibitor efficacy for RIP activity at physiologic pH has been previously limited by the pH 4.0 activity used for development of ricin A-chain transition state analogues. Ricin A-chain and other type I and II RIPs have a low pH catalytic optimum on nucleic acid substrates such as stem-loop RNA, poly(A) and/or hsDNA, while the natural ribosome substrate is depurinated optimally at physiologic pH (6, 18, 34). Saporin-S6 has a substrate specificity distinct from saporin-L1 and was not inhibited by saporin-L1 transition state mimics at neutral pH. Saporin-L1 rapidly catalyzes depurination of stem-loop, circular, and linear truncated mimics of the sarcin-ricin loop at neutral pH, a unique feature in the RIP family of N-glycohydrolases (Table 2) (35). Although saporin-L1 is reported to catalyze adenine release from poly(A), hsDNA, tRNA, E. coli rRNA, and globin mRNA at pH 7.8 (5, 6), the 80S ribosome is a preferred substrate.
Tight-binding inhibitors of saporin-L1 that prevent ribosome damage at physiological pH provide a breakthrough in developing immunotoxin cancer therapy. It should be possible to use saporin-L1 conjugates to target cancer cells. Following tumor lysis, rescue of the organism from vascular leak syndrome might be affected with the small, stable, and tight-binding inhibitors of saporin-L1 characterized here.
This application claims the benefit of U.S. Provisional Patent Application No. 61/338,376, filed on Feb. 17, 2010, the content of which is herein incorporated by reference.
This invention was made with government support under grant number CA 072444 awarded by the National Institutes of Health, U.S. Department of Health and Human Services. The government has certain rights in the invention.
| Number | Name | Date | Kind |
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| 5736536 | Siegall et al. | Apr 1998 | A |
| 7777025 | Schramm et al. | Aug 2010 | B2 |
| 20110053146 | Schramm et al. | Mar 2011 | A1 |
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| WO 2008105796 | Sep 2008 | WO |
| WO 2010053498 | May 2010 | WO |
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| Number | Date | Country | |
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| 20110201674 A1 | Aug 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61338376 | Feb 2010 | US |
