Roger A. Johnson

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.

Abstract Some properties of particulate and detergent-dispersed preparations of adenylate cyclase from rat brain have been studied. The activity of the particulate enzyme was greater with Mn2+ than with Mg2+ and the activity in the presence of either cation was enhanced by F- or nonionic detergents. In the presence of the non-ionic detergent Lubrol-PX, enzyme activity was further enhanced by dithiothreitol but inhibited by F-. Adenylate cyclase activity was dispersed into a 27,000 x g for 20 min supernatant fraction by Lubrol-PX. Inclusion of dithiothreitol in the homogenizing medium facilitated the solubilization of enzyme activity. More total activity was extracted from cerebellum than from cerebrum. The activity of the dispersed enzyme was essentially unchanged by filtration (0.22-µm pore) or by centrifugation at 100,000 x g for 1 hour. Separation of the detergent from the enzyme by gel filtration resulted in a turbid fraction, the activity of which was increased by F- or Lubrol-PX. Detergent-dispersed adenylate cyclase was inhibited by Ca2+, the degree of inhibition being dependent on whether Mg2+ or Mn2+ was present. In the presence of 8 mm Mg2+, ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) markedly decreased enzyme activity. The concentration causing 50% inhibition was dependent on protein concentration, but was usually about 30 µm. The inhibition by EGTA was completely prevented by the addition of an equimolar concentration of Ca2+, Mn2+, or Co2+, and was partially prevented by several other divalent cations. Gel chromatographic removal of EGTA from an inhibited enzyme completely restored activity. EDTA and several other metal-binding agents, at concentrations up to 1 mm, had little or no effect on adenylate cyclase activity. EGTA had no appreciable effect on either ATPase or cyclic 3':5'-nucleotide phosphodiesterase activities from brain or on adenylate cyclase activity prepared from partially purified plasma membranes from liver. The data suggest that adenylate cyclase from brain requires some metal in addition to added Mg2+ for full expression of activity and that the metal is bound tightly to the enzyme.

Natural products have historically been a mainstay source of anticancer drugs, but in the 90's they fell out of favor in pharmaceutical companies with the emergence of targeted therapies, which rely on antibodies or small synthetic molecules identified by high throughput screening. Although targeted therapies greatly improved the treatment of a few cancers, the benefit has remained disappointing for many solid tumors, which revitalized the interest in natural products. With the approval of rapamycin in 2007, 12 novel natural product derivatives have been brought to market. The present review describes the discovery and development of these new anticancer drugs and highlights the peculiarities of natural product and new trends in this exciting field of drug discovery.