Synthesis of novel 5-arylidenethiazolidinones with apoptotic properties via a three component reaction using piperidine as a bifunctional reagent.

The synthesis of a new library of 5-arylidenethiazolidinone compounds using an efficient three component reaction with thiazolidine-2,4-dione, piperidine and appropriate aldehydes is reported. This reaction is excellently high yielding, tolerant towards a variety of aldehydes and provides access to these compounds in a single step (in comparison to low yielding multistep syntheses reported in the literature). Once the reaction is complete, the desired product precipitates out of the reaction mixture and is isolated by filtration and purified by washing and recrystallization. These compounds revealed anti-proliferative activities against human breast cancer cells (MCF7 and MDA). Phenotypic profiling established the most active compound 17i (EC50 = 4.52 µM) as an apoptotic agent. A novel chemical proteomics approach identified ß-actin-like protein 2, γ-enolase and macrophage migration inhibitory factor (MMIF) as putative cellular binding partners of 17i.

NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by ß-lapachone.

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD(+) synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD(+) pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, ß-lapachone (ß-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. ß-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD(+) consumption. Synergy with FK866+ß-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD(+) synthesis while increasing NAD(+) consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)(+) depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, µ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD(+)-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD(+) synthesis and ß-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD(+) pools in cancer cells to sensitize them to ROS-mediated cell death by ß-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.

The enteric toxins of Clostridium perfringens.

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (approximately 25-35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, epsilon-toxin (a bioterrorism select agent) and beta-toxin, cause veterinary enterotoxemias when absorbed from the intestines; beta- and epsilon-toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, beta2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

Nucleoside triphosphate specificity of tubulin.

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.

Suppression of microtubule dynamic instability and treadmilling by deuterium oxide.

Deuterium oxide (D(2)O) is known to promote the assembly of tubulin into microtubules in vitro, to increase the volume of mitotic spindles and the number and length of spindle microtubules, and to inhibit mitosis. Reasoning that its actions on cellular microtubules could be due to modulation of microtubule dynamics, we examined the effects of replacing H(2)O with D(2)O on microtubule dynamic instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H(2)O with D(2)O promoted microtubule polymerization and stabilized microtubules against dilution-induced disassembly. Using steady-state axoneme-seeded microtubules composed of pure tubulin and video microscopy, we found that 84% D(2)O decreased the catastrophe frequency by 89%, the shortening rate by 80%, the growing rate by 50%, and the dynamicity by 93%. Sixty percent D(2)O decreased the treadmilling rate of microtubules composed of tubulin and microtubule-associated proteins by 42%, and 89% D(2)O decreased the steady-state GTP hydrolysis rate by 90%. The mechanism responsible for the ability of D(2)O to stabilize microtubule dynamics may involve enhancement of hydrophobic interactions in the microtubule lattice and/or the substitution of deuterium bonds for hydrogen bonds.

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen.

BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.

Indolylquinoline derivatives are cytotoxic to Leishmania donovani promastigotes and amastigotes in vitro and are effective in treating murine visceral leishmaniasis.

A wide variety of biologically active compounds contain indole and quinoline nuclei. Some novel indolylquinoline derivatives were synthesized from indole by Friedel-Crafts acylation reaction. Out of the four derivatives tested, 2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline) (C) had no effect on the promastigotes or amastigotes of Leishmania donovani in vitro. The remaining three analogues, 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D), inhibited the growth of L. donovani promastigotes in vitro and were cytotoxic to both the promastigote and amastigote forms of the parasite. These three derivatives were also effective in eliminating L. donovani amastigotes from BALB/c mouse peritoneal macrophages in vitro. One indolylquinoline derivative [A] was used to treat established visceral leishmaniasis in BALB/c mice. This compound was significantly more effective than sodium antimony gluconate (SAG) in reducing the splenic parasite load at a much lower concentration (5% of SAG). Our results suggest that indolylquinoline derivatives may be exploited as antileishmanial agents.

Stabilization of tubulin by deuterium oxide.

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.

Leishmania donovani parasites interact with gamma/delta+ human peripheral blood T cells and induce susceptibility to NK cell-mediated lysis.

We recently reported that Leishmania donovani infect the human T-cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of gamma/delta+ T cells was markedly increased when in vitro generated normal human T-cell blasts were cultured with L. donovani amastigotes. About 30% of the gamma/delta+ T cells in the parasite exposed T-cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T-cell blasts had a reduced surface expression of HLA-DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T-cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell-mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala-azar patients tested expressed intracellular L. donovani antigens.

Neuromuscular transmission and innervation in the urinary bladder of the insectivore Suncus murinus.

In isolated preparations of the urinary bladder detrusor of the house musk shrew Suncus murinus (order: insectivora; family: Soricidae), electrical field stimulation (0.5-32 pulses/s) evoked neurogenic contractile responses that were markedly attenuated by atropine (1 microM). The non-cholinergic component was reduced but not abolished by the P2-purinoceptor antagonist, suramin (300 microM). Thus, neuromuscular transmission in the suncus urinary bladder is effected by cholinergic and purinergic nerves together with an as-yet unidentified component. Using immunohistochemical methods, the suncus urinary bladder was seen to be supplied by nerves containing neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, galanin, substance P, calcitonin gene-related peptide and type I nitric oxide synthase. The pattern of responses to electrical field stimulation was more similar to that of humans and Old World primates, than to that of rodents or lagomorphs. The pattern of innervation of the bladder wall, in terms of the distribution of populations containing a given neuropeptide, was very similar to that in humans. Hence, Suncus murinus may provide a novel species for modelling the neuropharmacology of the human bladder, and also for studying the evolution of autonomic innervation.

Colchicine binding to tubulin monomers: a mechanistic study.

The kinetic and thermodynamic parameters for colchicine-tubulin and deacetamidocolchicine-tubulin interaction, under the condition where tubulin is predominantly in its dissociated state (approximately 80% monomer), have been determined. We observe that the kinetic parameters exihibit marked change when colchicine interacts with the monomeric form of tubulin rather than with the dimeric form of tubulin. The reaction of colchicine with tubulin monomers is characterized by an enhanced association rate which is a consequence of the lowering of activation energy. Colchicine-tubulin interaction, which is only poorly reversible, becomes partially reversible under this condition. Differences were also noticed in the thermodynamic parameters: the reaction of colchicine with tubulin monomers is enthalpy driven with small positive entropy, while with tubulin dimers a large positive entropy change was reported. However, no such changes in the binding parameters were observed for the reaction involving deacetamidocolchicine (a colchicine analog devoid of a side chain at the C-7 position of B-ring) with tubulin monomers. We therefore conclude that a single subunit of tubulin is capable of binding colchicine and that the unusual properties of colchicine-tubulin interactions such as the slow association rate, high activation energy, and the poor reversibility are due to the possible contact(s) of the C-7 substituent (in the B-ring) of colchicine with the other subunit of tubulin.

Peripheral blood mononuclear cells of patients with Indian visceral leishmaniasis suppress natural killer cell activity in vitro.

Peripheral blood mononuclear cells (PBMC) of patients with Indian visceral leishmaniasis (VL) suppressed the natural killer cell (NK) activity of normal human PBMC in vitro in a dose-dependent manner. Adherent monocytes, but not the non-adherent lymphocytes of VL patients suppressed NK activity through soluble mediators. The level of suppression seen in different patients was variable.

Thermodynamics of colchicinoid-tubulin interactions. Rrol of B-ring and C-7 substituent.

The quenching of tryptophan fluorescence has been used to determine the kinetic and thermodynamic parameters of binding of B-ring analogs of colchicine to tubulin. The on rate, activation energy, off-rate, and thermodynamics of binding reaction have been found to be controlled at different points of analog structure. The on-rate and off-rate of deacetamidocolchicine (DAAC) binding with tubulin is 17 times slower than that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone-tubulin (AC-tubulin) interaction, although both reactions have very similar activation energies. The presence of B-ring alone does not significantly affect the thermodynamics of the binding reactions either, since both AC-tubulin and DAAC-tubulin interactions are enthalpy driven. Introduction of a NH2 group at C-7 position of the B-ring, as in deacetylcolchicine (NH2-DAAC) lowers the on-rate further with a significant rise in the value of the activation energy. However, bulkier substitutions at the same position, as in demecolcine (NHMe-DAAC) and N-methyldemecolcine (NMe2-DAAC) have no significant additional effect either on the on-rate or on the value of activation energy. Introduction of NH2 group in the C-7 position of B-ring also increases the positive entropy of the binding reaction to a significant extent, and it is maximum when NMe2 is substituted instead of NH2 group. Thus, interaction of NH2-DAAC, NHMe-DAAC, and NMe2-DAAC with tubulin are entropy driven. Our results suggest that the B-ring side chain of aminocolchicinoids makes contact(s) with dimeric tubulin molecules.

Interaction of a fluorescent analog of N-deacetyl-N-methyl-colchicine (colcemid) with liver alcohol dehydrogenase.

The evidence for specific binding of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)--colcemid (NBD-colcemid), a fluorescent analog of colcemid (N-deacetyl-N-methyl-colchicine), to liver alcohol dehydrogenase is presented. Alcohol dehydrogenase bound NBD-colcemid in a time-dependent manner, enhanced the fluorescence intensity, and caused a large blue shift of the emission maximum of the free drug. The specificity of binding was determined for both the colchicine nucleus and the NBD moiety. The binding was not affected by the presence of alcohol or NAD in the reaction mixture. Preincubation of horse liver alcohol dehydrogenase with colcemid inhibited the binding to a considerable extent. NBD-colcemid inhibited the enzymic activity of alcohol dehydrogenase in a mixed-type noncompetitive mode with a Ki value of 32 microM, whereas colcemid showed noncompetitive inhibition with a Ki of 100 microM. The association rate constant of NBD-colcemid binding with liver alcohol dehydrogenase was 587 M-1 s-1 at 25 degrees C. The stoichiometry and dissociation constant of the binding reaction were 0.62/dimer and 12 microM, respectively. Donor quenching experiments showed that both tryptophans of alcohol dehydrogenase transferred energy to the bound NBD-colcemid. Thus, this study reports the binding of a colchicine analog to a protein other than tubulin with high affinity. It is concluded that NBD-colcemid binding to dehydrogenases is a general phenomenon, but the common structural element(s) that is responsible for the binding activity, and which exists among tubulin and dehydrogenases, has yet to be determined.

Impairment of natural killer cell activity in Indian kala-azar: restoration of activity by interleukin 2 but not by alpha or gamma interferon.

Indian kala-azar patients have normal numbers of peripheral blood NK cells but impaired functional activity due to decreased binding and lysis of target cells. This impairment of NK activity could not be corrected by exogenous recombinant human alpha or gamma interferon. However, recombinant human interleukin 2 was able to restore this activity by augmenting conjugate formation and lysis of target cells.

Treatment of small bowel diarrhea with electrolyte glucose drink.

PIP: The results of 102 cases treated with an oral electrolyte-glucose solution for rehydration caused by mild cases of small bowel diarrhea without using an antimicrobial agent in conjunction are presented. Clinical features, such as frequency of loose bowel movement, age distributions, and other relevant symptomatology are provided tabularly. The solution used consisted of: sodium chloride, .85 gm; potassium bicarbonate, 1 gm.; glucose, 17.5 gm.; boiled and cooled water, 500 ml. 97 of 102 were treated only with the oral electrolyte-glucose solution, and the remainder received intravenous fluid before initiation of oral rehydration. Due to follow-up problems, 13 cases were omitted from the statistical analysis; of the remaining 89, 84 were controlled within 72 hours (as judged by cessation of loose bowel movements). During therapy, breastfeeding or cow's milk was expressly forbidden, but 4 of the 5 failures were later discovered to have recieved breastfeedings, and 1 was marasmic. The treatment of small diarrhea, not having persistent vomiting or shock, with some suitable oral electrolyte-glucose solution only is highly successful, safe, and inexpensive. Success rate was 94.38%.^ieng