Emily R. Derbyshire

Structure and Regulation of Soluble Guanylate Cyclase

Nitric oxide (NO) is an essential signaling molecule in biological systems. In mammals, the diatomic gas is critical to the cyclic guanosine monophosphate (cGMP) pathway as it functions as the primary activator of soluble guanylate cyclase (sGC). NO is synthesized from l-arginine and oxygen (O(2)) by the enzyme nitric oxide synthase (NOS). Once produced, NO rapidly diffuses across cell membranes and binds to the heme cofactor of sGC. sGC forms a stable complex with NO and carbon monoxide (CO), but not with O(2). The binding of NO to sGC leads to significant increases in cGMP levels. The second messenger then directly modulates phosphodiesterases (PDEs), ion-gated channels, or cGMP-dependent protein kinases to regulate physiological functions, including vasodilation, platelet aggregation, and neurotransmission. Many studies are focused on elucidating the molecular mechanism of sGC activation and deactivation with a goal of therapeutic intervention in diseases involving the NO/cGMP-signaling pathway. This review summarizes the current understanding of sGC structure and regulation as well as recent developments in NO signaling.

Biochemistry of Soluble Guanylate Cyclase

Nitric oxide (NO) functions in biology as both a critical cytotoxic agent and an essential signaling molecule. The toxicity of the diatomic gas has long been accepted; however, it was not known to be a signaling molecule until it was identified as the endothelium-derived relaxing factor (EDRF). Since this discovery, the physiological signaling pathways that are regulated by NO have been the focus of numerous studies. Many of the cellular responses that NO modulates are mediated by the heme protein soluble guanylate cyclase (sGC). NO binds to sGC at a diffusion controlled rate, and leads to a several 100-fold increase in the synthesis of the second messenger cGMP from GTP. Other diatomic gases either do not bind (dioxygen), or do not significantly activate (carbon monoxide) sGC. This provides selectivity and efficiency for NO even in an aerobic environment, which is critical due to the high reactivity of NO. Several biochemical studies have focused on elucidating the mechanism of NO activation and O(2) discrimination. Significant advances in our understanding of these topics have occurred with the identification and characterization of the sGC-like homologues termed Heme-Nitric oxide and OXygen binding (H-NOX) proteins.

A nitric oxide/cysteine interaction mediates the activation of soluble guanylate cyclase

Nitric oxide (NO) regulates a number of essential physiological processes by activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. The mechanism of NO sensing was previously thought to result exclusively from NO binding to the sGC heme; however, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation. Furthermore, thiol oxidation of sGC cysteines results in the loss of enzyme activity. Herein the role of cysteines in NO-stimulated sGC activity investigated. We find that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking a non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway.

Liver-stage malaria parasites vulnerable to diverse chemical scaffolds

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite’s life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

BackgroundSoluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.ResultsWe have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule.ConclusionComparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

Dissociation of Nitric Oxide from Soluble Guanylate Cyclase and Heme-Nitric Oxide/Oxygen Binding Domain Constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length α1β1 sGC, and the constructs β1(1–194), β1(1–385), and β2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5′-[(α,β-methylene]triphosphate (GMPCPP) or the activator 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and β2(1–217) at 37 °C and dramatically accelerated NO dissociation from sGC at 10 °C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 °C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity “closed” form, which releases NO slowly, and a higher activity “open” form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors

Antimalarial drugs have thus far been chiefly derived from two sources—natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

The Next Opportunity in Anti-Malaria Drug Discovery: The Liver Stage

Malaria afflicts 350–500 million people annually, and this debilitating and deadly infectious disease exacts a heavy toll on susceptible populations around the globe. Efforts to find effective, safe, and low-cost drugs for malaria have sharply increased in recent years. Almost all of these efforts have focused on the cyclic blood stage of the disease, partly because the parasites can be easily maintained in culture through addition of human red blood cells to the growth medium, and partly because blood stage infection causes malarias characteristic symptoms. However, the asymptomatic liver stage, which the parasite goes through only once in its life history, presents the best opportunity for developing drugs that both hit new targets and also could be used in highly desirable eradication campaigns. Recent research, especially on the frequency of differentially expressed genes in blood and liver stage parasites, supports the feasibility of discovering stage-specific drugs. Discovering these drugs will require a high-throughput liver stage phenotypic screen comparable to the existing blood stage screens, and the basic tools for such a screen have recently been created.

Antibiotic and antimalarial quinones from fungus-growing ant-associated Pseudonocardia sp.

Three new members of the angucycline class of antibiotics, pseudonocardones A–C (1–3), along with the known antibiotics 6-deoxy-8-O-methylrabelomycin (4) and X-14881 E (5) have been isolated from the culture of a Pseudonocardia strain associated with the fungus-growing ant Apterostigma dentigerum. Compounds 4 and 5 showed antibiotic activity against Bacillus subtilis 3610 and liver-stage Plasmodium berghei, while 1–3 were inactive or only weakly active in a variety of biological assays. Compound 5 also showed moderate cytotoxicity against HepG2 cells.

Identification and Validation of Tetracyclic Benzothiazepines as Plasmodium falciparum Cytochrome bc1 Inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.