Raghavan Balachandran

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.

Structure–activity and High‐content Imaging Analyses of Novel Tubulysins

The synthesis and biological evaluation of three tubulysin analogs provides the first structure–activity relationship in this family of potent cytotoxic myxobacteria metabolites. Most importantly, the labile N,O‐acetal at N14 is not essential for biological activity. Further, structural simplifications are possible without abolishing biological activities. The N‐terminal amino acid can be replaced with N‐methylsarcosine, and the configuration at the acetoxy‐bearing stereocenter at C11 is important but not critical for almost all aspects of the biological profile. Our data encourage further development of these compounds as potential therapeutic agents in cancer treatment.

Total synthesis and biological evaluation of pederin, psymberin, and highly potent analogs.

The potent cytotoxins pederin and psymberin have been prepared through concise synthetic routes (10 and 14 steps in the longest linear sequences, respectively) that proceed via a late-stage multicomponent approach to construct the N-acyl aminal linkages. This route allowed for the facile preparation of a number of analogs that were designed to explore the importance of the alkoxy group in the N-acyl aminal and functional groups in the two major subunits on biological activity. These analogs, including a pederin/psymberin chimera, were analyzed for their growth inhibitory effects, revealing several new potent cytotoxins and leading to postulates regarding the molecular conformational and hydrogen bonding patterns that are required for biological activity. Second generation analogs have been prepared based on the results of the initial assays and a structure-based model for the binding of these compounds to the ribosome. The growth inhibitory properties of these compounds are reported. These studies show the profound role that organic chemistry in general and specifically late-stage multicomponent reactions can play in the development of unique and potent effectors for biological responses.

The potent microtubule-stabilizing agent (+)-discodermolide induces apoptosis in human breast carcinoma cells--preliminary comparisons to paclitaxel.

(+)–Discodermolide, a sponge–derived natural product, stabilizes microtubules more potently than paclitaxel despite the lack of any obvious structural similarities between the drugs. It competitively inhibits the binding of paclitaxel to tubulin polymers, hypernucleates microtubule assembly more potently than paclitaxel, and inhibits the growth of paclitaxel–reslstant ovarian and colon carcinoma cells. Because paclitaxel shows clinical promise for breast cancer treatment, its effects in a series of human breast cancer cells were compared to those of (+)–discodermolide. Growth inhibition, cell and nuclear morphological, and electrophoretic and flow cytometric analyses were performed on (+)–discodermolide–treated MCF–7 and MDAMB231 cells. (–f)–Discodermolide potently inhibited the growth of both cell types (IC50<2.5 nM) at concentrations similar to those observed with paclitaxel. Complete inhibition of growth occurred with 10 nM or greater of each drug and was not reversed by removal. (+)–Discodermolide–treated cells exhibited condensed and highly fragmented nuclei. Flow cytometric comparison of cells treated with either drug at 10 nM, a concentration well below that achieved clinically with paclitaxel, showed both caused cell cycle perturbation and induction of a hypodiploid cell population. (+)–Discodermolide caused these effects more extensively and at earlier time points. The timing and type of high molecular weight DNA fragmentation induced by the two agents was consistent with induction of apoptosis. The results suggest that (+)–discodermolide has promise as a new chemotherapeutic agent against breast and other cancers.

Streamlined Syntheses of (−)-Dictyostatin, 16-Desmethyl-25,26-dihydrodictyostatin, and 6-epi-16-Desmethyl-25,26-dihydrodictyostatin

The dictyostatins are a promising class of potential anti-cancer drugs because they are powerful microtubule-stabilizing agents, but the complexity of their chemical structures is a severe impediment to their further development. On the basis of both synthetic and medicinal chemistry analyses, 16-desmethyl-25,26-dihydrodictyostatin and its C6 epimer were chosen as potentially potent yet accessible dictyostatin analogues, and three new syntheses were developed. A relatively classical synthesis involving vinyllithium addition and macrocyclization gave way to a newer and more practical approach based on esterification and ring-closing metathesis reaction. Finally, aspects of these two approaches were combined to provide a third new synthesis based on esterification and Nozaki-Hiyama-Kishi reaction. This was used to prepare the target dihydro analogues and the natural product. All of the syntheses are streamlined because of their high convergency. The work provided several new analogues of dictyostatin, including a truncated macrolactone and a C10 E-alkene, which were 400- and 50-fold less active than (-)-dictyostatin, respectively. In contrast, the targeted 16-desmethyl-25,26-dihydrodictyostatin analogues retained almost complete activity in preliminary biological assays.

Altered levels and regulation of stathmin in paclitaxel-resistant ovarian cancer cells

Two paclitaxel(Ptx)-resistant ovarian cancer cell lines, 1A9/Ptx-10 and 1A9/Ptx-22, isolated from the 1A9 cell line (a clone of the A2780 line) by continuous exposure to Ptx and verapamil, have point mutations in their major β-tubulin gene and in one or both alleles of their TP53 gene. These cells were examined for alterations in cell cycle regulators and the tubulin-binding protein stathmin. Unlike parental cells, neither 1A9/Ptx-10 nor 1A9/Ptx-22 expressed detectable levels of p21WAF1/Cip1, a putative transcriptional regulator of stathmin, but did overexpress stathmin and Bcl2. No differences were noted in the expression levels of proliferative cell nuclear antigen or tyrosine-phosphorylated p34Cdc2. Ptx treatment altered little the expression of stathmin in the parental cell line, although it increased p21WAF1/Cip1 levels several-fold. Infection of Ptx-resistant lines with a wild-type TP53-bearing adenovirus (AdWTp53) changed cell cycle distribution and increased the levels of p21WAF1/Cip1, but caused no changes in stathmin levels. Microtubule drug resistance in ovarian carcinoma may be associated with altered p53/21WAF1/Cip1 regulatory pathways for stathmin expression and function.

Synthesis and biological assessment of simplified analogues of the potent microtubule stabilizer (+)-discodermolide

An efficient, convergent and stereocontrolled synthesis of simplified analogues of the potent antimitotic agent (+)-discodermolide has been achieved and several small libraries have been prepared. In all the libraries, the discodermolide methyl groups at C14 and C16 and the C7 hydroxy group were removed and the lactone was replaced by simple esters. Other modifications introduced in each series of analogues were related to C11, C17 and C19 of the natural product. Key elements of the synthetic strategy included (a) elaboration of the main subunits from a common intermediate and (b) fragment couplings using Wittig reactions to install the (Z)-olefins. Library components were analyzed for microtubule-stabilizing actions in vitro, for displacement of [3H]paclitaxel from its binding site on tubulin, for antiproliferative activity against human carcinoma cells, and for cell signaling and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even significant structural simplification can lead to analogues with actions related to microtubule targeting.

High-Content Analysis of Cancer-Cell-Specific Apoptosis and Inhibition of in Vivo Angiogenesis by Synthetic (−)-Pironetin and Analogs

The natural product (−)‐pironetin is a structurally simple small molecule microtubule‐perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic α,β‐unsaturated lactone moiety. We used alkaloid‐catalyzed acyl halide‐aldehyde cyclocondensation reactions in asymmetric total syntheses of (−)‐pironetin and three synthetic analogs, and evaluated their biological activities by high‐content analysis in cell culture and in a zebrafish model. Synthetic (−)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi‐drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (−)‐pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide‐aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (−)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule‐perturbing agents in the context of a whole organism or under conditions of multi‐drug resistance.

Cell-Based and Biochemical Structure-Activity Analyses of Analogs of the Microtubule Stabilizer Dictyostatin

Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G2/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (Ki) determinations using [14C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [14C]epothilone B with Ki values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (Ki values of 4.55 and 4.47 μM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of β-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.