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p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

PDF file - 43K, Effect of LY2228820 on MK2 phosphorylation in mouse peripheral blood mononuclear cells (PBMC) and from patients with multiple myeloma.

PDF file - 86K, Supplemental Table 1. Kinases where LY2228820 shows >1000-fold selectivity in vitro (p38 MAPK vs. other kinase).

PDF file - 90K, shRNA silencing of p38a MAPK in U-87MG glioma (Westerns and xenograft tumor growth data).

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of advanced breast cancers. While there is one PI3Kα inhibitor FDA-approved for patients with PI3Kα-mutated breast cancer, and many others in clinical development, all of these agents inhibit wild-type PI3Kα and its mutated form with approximate equal potency. As a result, their efficacy is limited by toxicities associated with on target wild-type PI3Kα inhibition, notably hyperglycemia as well as cutaneous and GI toxicity. LOX-22783 is a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor. Here, we describe the preclinical profile of LOX-22783. H1047R selectivity was measured using biochemical kinase activity and cell-titer Glo and signal transduction assays. Tumor growth inhibition, pharmacokinetic and pharmacodynamic effects were assessed in in vivo studies using xenograft and patient-derived xenograft (PDX)-models. LOX-22783 inhibited growth and signaling responses in multiple H1047R-driven breast cancer cell lines and demonstrated high selectivity for H1047R mutated PI3Kα (EC50 values 250 nM) as well as the other wild-type PI3K isoforms (beta, gamma, and delta, all EC50 >250nM). In enzyme and cell-based assays, LOX-22783 dissociated from PI3Kα H1047R at a slower rate (3-6 hrs) compared to alpelisib (≤10 mins), potentially allowing for extended inhibition of PI3Kα H1047R by LOX-22783. LOX-22783 also normalized the EGF-stimulated membrane-localization of PI3Kα H1047R while alpelisib did not. LOX-22783 was highly kinome-selective when assayed at 3 µM, with no inhibitory activity on 17 lipid kinases or 374 protein kinases. In preclinical species, LOX-22783 demonstrated high oral bioavailability, including exposure in the CNS, a common site of metastases for patients with breast cancer. In vivo, LOX-22783 demonstrated dose-dependent tumor regression in H1047R breast cancer models without inducing hyperglycemia or other toxicities. Tumor pharmacodynamic analyses confirmed successful pathway inhibition. At doses resulting in 90% pathway inhibition, tumor regressions of ≥60% were observed. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients, without wild-type PI3Kα inhibition limiting target coverage for the H1047R mutant form. These data demonstrate that LOX-22783 potently and selectively inhibits mutant H1047R, but not wild-type PI3Kα, or other PI3K isoforms. LOX-22783 binds to an allosteric pocket distinct from the ATP binding site used by the approved and investigational PI3Kα inhibitors. We hypothesize that this profile will lead to differentiated efficacy and tolerability for patients with PI3Kα H1047R-mutated cancers, with the additional potential to address brain metastases. An IND submission is planned for 2022. Citation Format: Anke Klippel, Rui Wang, Loredana Puca, Andrew Lee Faber, Weihua Shen, Shripad V. Bhagwat, Kannan Karukurichi, Feiyu Fred Zhang, Carmen Perez, Ramon Rama, Ana Ramos, Yi Zheng, Zahid Bonday, James Thomas, Harold B. Brooks, Lisa J. Kindler, Sarah M. Bogner, Parisa Zolfaghari, Mark Hicks II, Sophie Callies, Brian Mattioni, Laurie LeBrun, Jim Durbin, Erin Anderson, Chris Mayne, Edward Kesicki, Gabrielle Kolakowski, Steven W. Andrews, Barbara J. Brandhuber. Preclinical characterization of LOX-22783, a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P142.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression. This work was supported by Eli Lilly and Company. We would like to thank the Genomics and Flow Cytometry units at the CNIO for technical help, C. Pantoja and the CNIO-Lilly Cell Signaling Therapies Laboratory for sharing protocols and reagents. Sí

Structure-based design approach was successfully used to guide the evolution of imidazopyridine scaffold yielding new structural class of highly selective inhibitors of cyclin dependent kinases that were able to form a new interaction with an identified residue of the protein, Lys89. Compounds from this series have shown no detectable effect when tested against a representative set of other serine/threonine kinases such as GSK3beta, CAMKII, PKA, PKC-alpha,beta,epsilon,gamma. Compound 2i inhibits proliferation in HCT 116 cells in tissue culture. Synthesis, co-crystal structure of CDK2 in complex with compound 2i, and preliminary SAR study are disclosed.

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.

The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good antiproliferative agents.

The synthesis and kinase inhibitory activity of a series of novel 1,7-annulated indolocarbazoles 6 and 16 is described. These compounds exhibited potent inhibitory activity against cyclin-dependent kinase 4 and good antiproliferative activity in a human colon carcinoma cell line.

The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.

A series of indolo[2,3-a]pyrrolo[3,4-c]carbazoles and their bis-indolylmaleimides precursors have been prepared in order to compare their activity as D1-CDK4 inhibitors. Both enzymatic and antiproliferative assays have shown that the structurally more constrained indolo[2,3-a]pyrrolo[3,4-c]carbazoles are consistently more active (8-42-fold) in head-to-head comparison with their bis-indolylmaleimides counterparts. Cell-cycle analysis using flow cytometry have also shown that the indolocarbazoles are selective G1 blockers while the bis-indolylmaleimides arrest cells in the G2/M phase.

The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.

A novel series of pyrrolo[3,4-c] carbazoles fused with a quinolinyl/isoquinolinyl moiety were synthesized and their D1/CDK4 inhibitory and antiproliferative activity were evaluated. Compound 8H, 14H-isoquinolinyl[6,5-a]-pyrrolo[3,4-c]carbazole-7,9-dione (1d) was found to be a highly potent D1/CDK4 inhibitor with an IC50 of 69 nM. Compound 1d also inhibited tumor cell growth, arrested tumor cells in G1 phase and inhibited pRb phosphorylation.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, alpha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 A resolution. The protein crystallizes in the space group P2(1) (a = 66.8 A, b = 154.5 A, c = 77.1 A, beta = 90.58 degrees ), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 A resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a beta/alpha-barrel, and the C-terminal domain of ODC is a modified Greek key beta-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27 degrees relative to each other. In addition, two of the beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of beta-strands and loops in the beta/alpha-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the delta-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

Ornithine decarboxylase (ODC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the first committed step in the biosynthesis of polyamines. The UV-visible spectra of PLP (300-500 nm) was used to monitor the formation and breakdown of ODC reaction intermediates by multiwavelength stopped-flow spectroscopy to determine the reaction mechanism. Global kinetic analysis of the spectral data acquired after mixing ODC with saturating substrate (S) or product (P) (10 mM ornithine or 10 mM putrescine at 4 degrees C) suggests that ODC-catalyzed decarboxylation proceeds by the following reaction mechanism: ODC + S if A --> B --> C --> D --> E/F if ODC + P, where A-F are intermediates along the reaction path. Species B, which has absorbance maxima of 350 and 450 nm, is spectrally distinct from the other intermediates. On the basis of the calculated spectral characteristics, species B is likely to represent a quinoid intermediate which would be formed directly upon decarboxylation of ornithine. Thus, the data suggest that the reaction proceeds via formation of a Schiff base intermediate (species A) during the dead time of the stopped-flow instrument, followed by formation of a quinoid intermediate with a rate constant of 21 s-1. The quinoid intermediate decays in two steps (with rates of 145 and 1.0 s-1, respectively) to a Schiff base with putrescine (species D). Protonation of the Calpha carbon is required for the formation of species D, suggesting that the first of these events represents this step. The decay of species D to free enzyme and product occurs via a minimum of two intermediates and at an overall rate constant of 1-3 s-1. By comparison to the steady-state turnover number (kcat = 0.5 s-1 at 4 degrees C), these data identify product release as a rate-determining step in the overall reaction.

Gliomas are the most devastating of primary adult malignant brain tumors. These tumors are highly infiltrative and can arise from cells with extensive self-renewal capability and chemoresistance, frequently termed glioma-propagating cells (GPCs). GPCs are thus the plausible culprits of tumor recurrence. Treatment strategies that eradicate GPCs will greatly improve disease outcome. Such findings support the use of GPCs as in vitro cellular systems for small-molecule screening. However, the nuances in using GPCs as a cellular screening platform are not trivial. These slow-growing cells are typically cultured as suspension, spheroid structures in serum-free condition supplemented with growth factors. Consequently, replenishment of growth factors throughout the screening period must occur to maintain cells in their undifferentiated state, as the more lineage-committed, differentiated cells are less tumorigenic. We present a case study of a small-molecule screen conducted with GPCs and explain how unique sphere activity assays were implemented to distinguish drug efficacies against the long-term, self-renewing fraction, as opposed to transient-amplifying progenitors, the latter of which are detected in conventional viability assays. We identified Polo-like kinase 1 as a regulator of GPC survival. Finally, we leveraged on public glioma databases to illustrate GPC contribution to disease progression and patient survival outcome.

We propose a new method that allows screening oncology drug combinations using data from in vitro studies to select agents that have the promise of showing a synergistic effect in vivo. In contrast to known approaches that define combination effects either on the concentration scale or on the percent inhibition scale, we use the growth rate of treated cells as a primary indicator of treatment activity. The developed method is based on a novel statistical model that describes the growth of cancer cells that are subject to treatment with a combination of compounds. The model assumes a multicompartment cell population with transition rates between compartments modeled according to biochemical reaction properties, and cells in each compartment growing according to exponential law. This translates to a linear system of ordinary differential equations, whose solution is accurately approximated by a closed-form expression using rapid equilibrium assumptions. Special cases of the aforementioned model represent situations when the combination effect is absent or when the considered drugs act as the same compound. Assuming the normal distribution for the growth rate measurement error, we describe a formal statistical testing procedure to distinguish between different mechanisms of action for the considered compounds, and to test if a significant combination effect is being observed.

Diaquacobinamide (DAC) was prepared by reducing aquacyanocobinamide (Factor B) with zinc and oxidising in aqueous HCl. The macroscopic acid dissociation constants p K Co1 and p K Co2 for coordinated H 2 O in DAC were found spectrophotometrically to be 5.91 ± 0.04 and 10.30 ±0.24 at 25 °C; from their temperature dependence it was found that Δ H =45±4 and 27±2 kJ mol −1 , and Δ S =40±14 and −109±8 J K −1 mol −1 , for p K Co1 and p K Co2 , respectively. Equilibrium constants for the reaction of the cobinamide with cyanide were determined in the pH range 8–12 at 25 °C, μ=1.0 M (NaClO 4 ). Two CN − ligands bind to aquahydroxocobinamide and pH dependence of the observed equilibrium constants can be accounted for by assuming that both dihydroxocobinamide and hydroxocyanocobinamide are inert, while equilibrium constants for reaction of the labile species aquahydroxocobinamide and aquacyanocobinamide with HCN are insignificantly small compared to those for reaction with CN − anion. A fit of the data shows that the macroscopic equilibrium constant, log β 2 , for reaction of 2CN − with the two diastereomers of aquahydroxocobinamide to produce dicyanocobinamide is 19.0±0.1. Hence the macroscopic log K for binding of CN − to the two diastereomers of aquahydroxocobinamide is 11 since log K for binding of CN − to aquacyanocobinamide is 8. Equilibrium constants for binding of azide, pyridine, N - methylimidazole and 3-amino-l-propanol to aquahydroxocobinamide were determined at 25 °C, μ=1.0 M (NaClO 4 ), pH 12. In these cases only a single ligand is bound; the macroscopic log K values are N -methylimidazole, 6.06±0.04; 3-amino-l-propanol, 4.66±0.04; pyridine, 4.19±0.01; and azide, 3.45±0.04. A comparison of the present results with others available shows that hydroxide is above water and 5, 6-dimethylbenzimidazole, but below cyanide, sulfite and alkyl ligands in the trans influence order of the cobalt corrinoids.

Binding of neopentylcobalamin and benzylcobalamin to the apoprotein of a haptocorrin from chicken serum has been demonstrated spectrophotometrically. The spectra of the protein-bound cobalamins strongly resemble those of base-on alkylcobalamins and show that when unbound these sterically hindered alkylcobalamins are only approximately 75% (benzyl) and 40% (neopentyl) base-on, at neutral pH and at 5 degrees C. The haptocorrin was found to stabilize the spontaneous thermal decomposition of the neutral species of benzylcobalamin and neopentylcobalamin by 470-fold (3.6 kcal) and 166-fold (3.0 kcal), respectively, relative to the protein-free species. After correction of the activation parameters for the thermal decomposition of the protein-free, neutral alkylcobalamins for the relative proportions of base-on and base-off species, the haptocorrin was found to stabilize the base-on species of both alkylcobalamins by 275- to 1400-fold (approximately 3.3 to 4.3 kcal). From the temperature dependence of the decomposition reactions, the enthalpies of activation are found to be essentially identical for the protein-free and protein-bound species of either cobalamin. Thus, stabilization of the thermal decomposition of these sterically hindered alkylcobalamins by haptocorrin is entirely due to entropic factors.

Spectroscopic and thermodynamic evidence for the structure of cobalamines and dicyanocobalamin (CN){sub 2}Cbl have been previously reported. The structure indicated the occurrence of the so-called tuck-in species. Further observations and characterization of the tuck-in species of (CN){sub 2}Cbl by {sup 15}N NMR spectroscopy are presented herein. These results represent the first observation of the {sup 15}N NMR spectrum of benzimidazole nucleotide of cobalamins. The first NMR observation of the amide protons of cobalamins and their connectivity to the amide nitrogens are also reported. 50 refs., 2 figs., 2 tabs.

The synthesis of a novel series of 1,7-annulated indolocarbazoles 2 and 16 is described. These compounds were found to be potent cyclin dependent kinase inhibitors with good antiproliferative activity against two human carcinoma cell lines. These inhibitors also arrested tumor cells at the G1 phase and inhibited pRb phosphorylation.

The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/CDK4.

Novel substituted indolocarbazoles were synthesized, and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles 4 were found to be potent and selective D1/CDK4 inhibitors. 4d and 4h exhibited potent and ATP-competitive D1/CDK4 activities with IC50 values of 76 and 42 nM, respectively. Both compounds had high selectivity against the other kinases. These D1/CDK4 inhibitors inhibited tumor cell growth, arrested tumor cells at the G1 phase, and inhibited pRb phosphorylation.

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.

Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff base with PLP in the absence of substrate. The mechanistic role of this residue (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by analysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC). Analysis of K69A ODC demonstrated that the enzyme copurified with amines (e.g. putrescine) that were tightly bound to the active site through a Schiff base with PLP. In contrast, on the basis of an absorption spectrum of K69R ODC, PLP is likely to be bound to this mutant enzyme in the aldehyde form. Pre-steady-state kinetic analysis of the reaction of K69R ODC with L-Orn and putrescine demonstrated that the rates of both the product release (k(off.Put) = 0.0041 s(-)(1)) and the decarboxylation (k(decarb) = 0.016 s(-)(1)) steps were decreased by10(4)-fold in comparison to wild-type ODC. Further, the rates of Schiff base formation between K69R ODC and either substrate or product have decreased by at least 10(3)-fold. Product release remains as the dominant rate-limiting step in the reaction (the steady-state parameters for K69R ODC are k(cat) = 0.0031 s(-)(1) and K(m) = 0.18 mM). The effect of mutating Lys-69 on the decarboxylation step suggests that Lys-69 may play a role in the proper positioning of the alpha-carboxylate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not mediate substrate specificity. Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines. Kinetic analysis of the binding of a series of diamines and amino acids to K69R ODC suggests that noncovalent interactions in the active site of K69R ODC promote selective ligand binding during Schiff base formation.

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A.,Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.

A circular dichroism (CD) assay for decarboxylation of optically pure amino acids is described. The viability of this assay is demonstrated using the Trypanosoma brucei ornithine decarboxylase (ODC)-catalyzed reaction of L-ornithine to putrescine and CO2. The results from the CD assay (kcat of 7.5 +/- 0.7 s-1 and Km 230 +/- 60 microM) were identical to the results obtained from the commercially available dye-linked assay which couples CO2 production with NADH oxidation (kcat of 7.3 +/- 0.5 s-1 and Km 320 +/- 30 microM). The CD assay has advantages over the currently used 14CO2 and dye-linked assays since it can be continuously monitored and does not contain additional enzymes. The CD assay will enable the determination of the effects of pH, ionic strength, and D2O on catalysis by ODC. Furthermore, the availability of cuvets with pathlengths from 0.01 to 100 mm provides an effective range for the CD assay from 10 microM to 2.5 M L-ornithine concentration for this assay. This technique should be generally applicable for steady-state analysis of other decarboxylases but is not easily amenable to the analysis of crude enzyme preparations.

The dependence of the second-order rate constants for replacement of H2O in aquahydroxocobinamide by azide at 25.0 °C, ionic strength l= 1.0 mol dm–3(KCl) in the range pH 9–12 showed that dihydroxocobinamide is inert to substitution. The kinetics of substitution of bound H2O in aquahydroxocobinamide by L = cyanide, azide, pyridine, N-methylimidazole or 3-aminopropan-1-ol was investigated as a function of ligand concentration and temperature by stopped-flow spectrophotometry at pH 12.0 and a constant l of 2.0 mol dm–3(except for 3-aminopropan-1-ol where l= 1.0 mol dm–3 because of the limited solubility of the ligand). The observed pseudo-first-order rate constants (corrected, where appropriate, for protonation of the N-donor atom of L, and the presence of inert dihydroxocobinamide) showed the onset of saturation with ligand concentration for all ligands, with the exception of 3-aminopropan-1-ol. The saturation effect proves that the reaction proceeds through a dissociative activation pathway. Furthermore, the observation that the saturation rate constant, ksat(and its activation parameters ΔH‡ and ΔS‡), depends on the identity of L indicates that incoming L participates in the transition state. This allows the mechanism of the reaction to be identified as a dissociative interchange.

In this article, Dr. Brooks discusses the what, the when, the where, the why, and the how of science education in the secondary school. He discusses various objectives that the good school administrator considers in setting up functional courses of study in science or in evaluating them. He shows how science courses must be kept in line with the time. He states "that the principal who would build well in the field of science instruction must select his objectives both general and specific with care; he must be sure that they are sound in terms of educational philosophy, community understanding, and pupil needs and interests."

in each of the above cobalamin analogues. These comparisons eliminate the b. d. and e amides as possible hydrogen-bond donors in the tuck-in species. Methylation of the benzimidazole B3 nitrogen was shown to prevent formation of the tuck-in species in the dicyano derivative of the trimethylbenzimidazolyl analogue by comparison of its I3C spectrum to that of the detached, N-methylated nucleotide methyl ester. Taken together with previous ISN NMR results, numerous NOE's observed between the benzimidazole B2, 84, and B7 protons and protons on the corrin side chains, ring and ring methyl groups strongly suggest that the g side-chain amide is the hydrogen-bond donor in the tuck-in species.