Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs.

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.

Intraoperative coronary artery dilatation with angioscopic guidance.

The use of intraoperative balloon dilatation during coronary artery bypass surgery has been limited due to relatively unpredictable and potentially damaging results. The development of fiberoptic angioscopy permits safe visualization of the interior of coronary arteries and may be a valuable adjunct to intraoperative balloon dilatation. A 56-year-old male underwent four vessel coronary grafting with progressive intraoperative balloon dilatation of a second more distal stenosis of the left anterior descending coronary artery. Angioscopy was used to determine optimal balloon sizing and allowed visualization of associated intimal changes that occurred during the procedure, resulting in a successful outcome for this patient.

Intraoperative angioscopy for coronary bypass surgery.

Despite advances in coronary artery surgery, technical abnormalities remain a significant cause of early graft closure. The development of small fiberoptic angioscopes now allows direct intravascular magnified examination. Seventy-five distal anastomoses and vein grafts, and five selected coronary arteries were examined with 0.8- to 2.5-mm diameter angioscopes introduced through the proximal vein graft while irrigating with clear cardioplegia. Angioscopic findings were correlated with angiographic data, vessel morphology, graft flow, and postoperative course. Satisfactory images were obtained in 72 of 75 anastomotic inspections. Each examination took less than 2 minutes and required less than 100 cc of flush. Angioscopic abnormalities that did not require revision were noted in 17 of 72 anastomoses; intimal flaps in 9, thrombus on posterior wall plaque in 4, intimal irregularities in 4, bucking of posterior wall in 3, and valve near anastomoses in 1. No outflow obstruction nor misplaced sutures were noted. Average flow rate through the grafts with anastomotic angioscopic abnormalities was 33 cc/min versus 40 cc/min in the remaining grafts. However, regression analysis revealed that low-graft flow was correlated with vessel size and runoff but was not with angioscopic findings. Intracoronary angioscopy revealed discrepancy with angiographic findings in 4 of the 5 examinations. No complications occurred as a result of angioscopy. No graft closure has occurred during early follow-up. Intraoperative angioscopy can be done with minimal alteration of the usual routine. The 24% occurrence of minor angioscopic abnormalities did not appear to compromise graft flow or early patency.

Complexities in the management of arterial compromise due to thoracic outlet syndrome.

A patient with arterial compromise secondary to thoracic outlet obstruction is presented and discussed. The case highlights the difficulty in management when initial surgery does not adequately decompress the outlet. It also demonstrates the risk to limb viability that may ensue.

Intraoperative coronary angioscopy: development of practical techniques.

Techniques and equipment for intraoperative coronary angioscopy were studied in the coronary arteries and cardiac veins of excised and in vivo animal hearts. These studies then allowed development of safe, practical techniques for human clinical use. In coronary artery bypass operations, multichannel angioscopes of 2.3 to 2.8 mm diameter gave the best results for examinations of the bypass vein and anastomosis, whereas smaller optical fibers of 1 mm diameter were required for inspection of the native coronary artery. Abnormalities were detected in 11 of 48 (23%) coronary bypass anastomoses, and significant discrepancy in the degree of coronary artery stenosis as compared with the preoperative angiogram was revealed in 2 patients. The authors concluded that a new design of ultrafine, multichannel angioscope would be more suitable to the different requirements for inspection of both the anastomosis and the recipient artery.

Bleeding Meckel's diverticulum in an adult.

The diagnosis of a symptomatic Meckel's diverticulum in an adult is uncommon. Still more infrequent is the presentation of a bleeding Meckel's diverticulum after childhood. We present a case report of a 24-year-old male with gastrointestinal hemorrhage secondary to a Meckel's diverticulum containing ectopic gastric mucosa. With the exception of a Meckel's Tc 99m pertechnetate scan, all other diagnostic procedures including Tc 99m-labeled red cell scan and angiography were negative.

Endoscopic intravascular surgery removes intraluminal flaps, dissections, and thrombus.

Over the last 3 years angioscopic techniques have been used to guide intraluminal instrumentation in 73 patients undergoing thrombectomy, nine patients with vascular trauma, and 32 patients during laser angioplasty and balloon dilation. After balloon-catheter thromboembolectomy residual, occlusive thrombi tightly adherent to the arterial wall were removed with flexible biopsy forceps in 13 of 73 (18%) patients; underlying intimal flaps were removed in another four. In nine patients traumatic intimal defects caused by iatrogenic cannulation injuries (n = 5) or external trauma (n = 4) were managed by thrombectomy followed by complete or partial intravascular removal of the intimal flap (n = 6) or dissection plane (n = 3) with long flexible forceps and rotating brushes. Traumatic intimal defects observed in two additional patients were judged to be too severe for endoscopic manipulation and required immediate bypass grafting. Inspection after angioplasty in 32 patients revealed wall charring and obvious thermal damage after laser procedures in 28 (87%) and plaque cracking, intimal flaps, and fragmentation in 26 (81%). These defects were underestimated on intraoperative angiography. Large flaps and thrombus were removed endoscopically in three. We conclude that angioscopic study reveals the extent of intimal injury and gives insights into mechanisms of instrumentation. Adherent thrombus after embolectomy by balloon catheter and intimal flaps caused by trauma or angioplasty are common and, if severe, can be successfully treated by endoscopic intravascular manipulation in selected patients.

Experimental and clinical applications of angioscopic guidance for laser angioplasty.

The role of angioscopic monitoring and aiming for control of laser intervention in the vascular system was initially investigated in 48 vessels in 33 dogs, and the techniques were then applied to 30 patients undergoing intraoperative or percutaneous laser-probe angioplasty treatment for long atherosclerotic occlusions of the femoral and popliteal arteries or well-localized lesions of the superficial femoral artery. Experimental bare argon fiber laser application in 20 normal canine arteries in vivo demonstrated that small-diameter laser fibers could be accurately aimed by manipulations of the scope. However, advancement of the fiber resulted universally in perforation, with extravasation and thermal damage of surrounding tissues after 2 seconds of argon laser energy at low power. In 28 canine and 2 human veins, angioscopically guided metallic-tipped laserprobes were used to divide 82 valve cusps in preparation for in-situ bypass, with satisfactory aiming and monitoring achieved expeditiously by manipulations of the angioscope. We conclude that angioscopic aiming of lasers is feasible in normal vessels or localized lesions. In contrast, angioscopy has a restricted role for guidance of laser angioplasty in atherosclerotic, occluded arteries, and does not prevent perforation. Postprocedural inspection allows immediate detection of complications and may avert or predict poor outcome.

Relative substrate activities of structurally related pteridine, quinazoline, and pyrimidine analogs for mouse liver folylpolyglutamate synthetase.

Several structurally related series of folate analogs were studied as substrates for mouse liver folylpolyglutamate synthetase (FPGS). A comparison of the kinetics of the interaction of this enzyme with folate analogs that contained the quinazoline ring in place of the pteridine ring with those of the analogous pteridines demonstrated that the quinazoline derivatives were more efficient substrates for and tighter binding inhibitors of this enzyme. A series of 2,4-diaminopyrimidine dihydrofolate reductase inhibitors were found to be substrates for FPGS; these are the first known compounds without a fused ring system analogous to the pteridine ring of the folate molecule that are substrates for FPGS. Several 5,8-dideazafolate derivatives that lack the 2-amino group had activity as substrates for FPGS equivalent to that of the corresponding 5,8-dideazafolates. When a homologous series of 5,8-dideazafolic acid analogs with hydrocarbon substituents on the 10-nitrogen were studied, these substituents were found to diminish the efficiency of utilization of these analogs as substrates for FPGS; this effect increased with increasing chain length of the hydrocarbon. It was concluded that neither the 2-amino group nor an intact pyrazine ring of folates and folate analogs are essential for the binding of folates to the active site of mouse liver FPGS but that the pyrazine ring probably serves to position other regions of the folate molecule that interact with amino acid residues in the active site. It was also inferred from these observations that the volume within the active site of FPGS above/below the pyrazine ring or near the 10-position of folate derivatives are regions of limited bulk tolerance; binding of folate analogs with substituents at these positions probably distorts the active site.

Evaluation of pteroyl-S-alkylhomocysteine sulfoximines as inhibitors of mammalian folylpolyglutamate synthetase.

The similarity between the reactions catalyzed by folylpoly-gamma-glutamate synthetase (FPGS), gamma-glutamylcysteine synthetase, and glutamine synthetase, as well as the susceptibility of the latter two enzymes to inhibition by methionine sulfoximine, suggest that folic acid derivatives with methionine sulfoximine or its alkyl homologs in place of the glutamate side chain of folate are good candidates to act as enzyme-generated transition state analog inhibitors of the FPGS reaction. Thus, pteroylmethionine sulfoximine, and the homologous S-ethyl-, S-propyl-, and S-butylhomocysteine sulfoximine derivatives were evaluated as inhibitors of FPGS that was partially purified from mouse liver and from mouse L1210 cells. The related compound, pteroyl-S-methylhomocysteine sulfone, which cannot undergo enzyme-mediated activation, was also investigated. Unexpectedly, none of these compounds showed significant inhibition of FPGS from these sources under a variety of conditions. These results, taken together with previously established structure-activity correlations, imply that a negative charge at the gamma-position of folate analogs may be required for initial binding to FPGS and thus constitutes a prerequisite for activity of potential mechanism-based inhibitors of this enzyme.

Structural requirements for the activity of antifolates as substrates for mammalian folylpolyglutamate synthetase.

The activity of mouse-liver folylpolyglutamate synthetase (FPGS) was compared using a number of folates and folate analogs in order to determine which structure modifications were compatible with enzyme catalysis and with efficient binding to enzyme. Most structural alterations in the amino acid side chain eliminated activity as a substrate for this enzyme, whereas modifications of any of several positions in the pteridine ring were tolerated with retention of FPGS substrate activity. Folate analogs with the lowest apparent Michaelis constants (Km,app) had a) a 4-amino group, b) a 5,6,7,8 reduced ring system, c) a quinazolate ring, and/or d) an unsubstituted 10-position. There was some correlation between FPGS substrate activity and the potency of folate antimetabolites as cytotoxic compounds but not necessarily as compounds selectively cytotoxic to tumor cells.

Methotrexate analogues-27. Dual inhibition of dihydrofolate reductase and folylpolyglutamate synthetase by methotrexate and aminopterin analogues with a gamma-phosphonate group in the side chain.

gamma-Phosphonate analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively, by reaction with methyl D,L-2-amino-4-phosphonobutyrate followed by gentle alkaline hydrolysis. The products were compared with the corresponding D,L-homocysteic acid derivatives as inhibitors of dihydrofolate reductase and folylpolyglutamate synthetase, and as inhibitors of cell growth in culture. The gamma-phosphonates were somewhat less active than either the gamma-sulfonates or the parent drugs as inhibitors of murine dihydrofolate reductase. The MTX gamma-sulfonate and gamma-phosphonate analogues were equally inhibitory toward mouse liver folylpolyglutamate synthetase (Ki = 190 microM), but in the AMT series the gamma-phosphonate (Ki = 8.4 microM) was more potent than the gamma-sulfonate (Ki = 45 microM). The AMT analogues were consistently more inhibitory than the MTX analogues against cultured L1210 murine leukemia cells, but neither the gamma-phosphonates nor the gamma-sulfonates were as potent as their respective parent drugs. The gamma-phosphonate analogue of MTX was three times more potent than MTX against the MTX-resistant mutant line L1210/R81, but the AMT gamma-phosphonate was less potent than AMT; however, these differences were small in comparison with the level of resistance to all these compounds in the L1210/R81 line. The results suggest that N10-methyl and N10-unsubstituted compounds altered at the gamma-position do not necessarily follow identical structure-activity patterns in every test system.

Structural features of 4-amino antifolates required for substrate activity with mammalian folylpolyglutamate synthetase.

The activity of a series of folic acid analogues as substrates for partially purified mouse liver folylpolyglutamate synthetase was determined and the effects of substituents on the binding to, and catalytic processes of, this enzyme were inferred. A 4-amino group improved substrate activity primarily by decreasing the apparent Km while N10-methyl substitution substantially diminished utilization as a substrate, again, by effects on Km. Isosteric replacement of N-10 altered substrate activity. A free alpha-carboxyl group in the amino acid side chain was required for catalysis as was the presence of the side chain amide carbonyl group. Modification of the amino acid side chain length profoundly affected activity. Several observations were made that may be relevant to chemotherapy with folate antimetabolites: 1) 7-hydroxymethotrexate was a substrate for this enzyme; 2) substrate activity and substrate inhibition were observed with CB 3717, a potent inhibitor of thymidylate synthase; 3) potent classical dihydrofolate reductase inhibitors were identified that were either not substrates for mouse liver folylpolyglutamate synthetase (e.g., 4-amino-4-deoxy-N10-methylpteroyl-L-alpha-aminoadipate) or were much better substrates than methotrexate for this enzyme (e.g., aminopterin); and 4) leucovorin and methotrexate appeared to be substrates for the same synthetase, but leucovorin saturated the reaction at much lower concentrations. These results have implications for the design of folylpolyglutamate synthetase inhibitors and for the selection of dihydrofolate reductase inhibitors that are either not polyglutamated or are efficiently polyglutamated in vivo.

A mechanism for the addition of multiple moles of glutamate by folylpolyglutamate synthetase.

The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme.

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.

Measurement of folylpolyglutamate synthetase in mammalian tissues.

Previous methods for the measurement of folylpolyglutamate synthetase have been modified and combined to facilitate assay of this enzyme at the levels found in mammalian tissues. Batch adsorption of product onto charcoal allowed the rapid analysis of multiple samples of partially purified enzyme, e.g., column fractions. This technique, however, was unsuitable for the assay of folylpolyglutamate synthetase in crude cytosols due to the presence of interfering enzyme activities. On the other hand, the sequential use of charcoal adsorption and batch elution from DEAE-cellulose permitted isolation of the folate product from assay mixtures containing crude enzyme fractions. Under these conditions, interference from other enzyme activities and background values were low enough for the quantitation of 10 pmol of oligoglutamyl folate product. Folylpolyglutamate synthetase was measured in a series of mouse tissues and tumors. Enzyme activity was quite low in all cases. Mouse liver and kidney and some of the tumors studied had the highest levels (50-100 pmol product/h/mg protein); other tumors and spleen had lower levels. Enzyme activity was at the limit of detection in intestine and lung and was below detection in brain, heart, and skeletal muscle.