Scaffold Hopping and Optimization of Small Molecule Soluble Adenyl Cyclase Inhibitors Led by Free Energy Perturbation.

Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.

Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum.

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light-induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the excessively absorbed energy. Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light-induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin-binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization.

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

Metabolic changes in mouse sperm during capacitation†.

Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.

Optimization of lead compounds into on-demand, nonhormonal contraceptives: leveraging a public-private drug discovery institute collaboration†.

Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.

Capacitation increases glucose consumption in murine sperm.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.

Distinct intracellular sAC-cAMP domains regulate ER Ca2+ signaling and OXPHOS function.

cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increasing OXPHOS proteins. Importantly, sAC knockout cells also exhibit decreased probability of endoplasmic reticulum (ER) Ca2+ release associated with diminished phosphorylation of the inositol 3-phosphate receptor. Restoring sAC expression exclusively in the mitochondrial matrix rescues OXPHOS activity and reduces mitochondrial biogenesis, indicating that these phenotypes are regulated by intramitochondrial sAC. In contrast, Ca2+ release from the ER is only rescued when sAC expression is restored throughout the cell. Thus, we show that functionally distinct, sAC-defined, intracellular cAMP signaling domains regulate metabolism and Ca2+ signaling.

Physiological roles of acid-base sensors.

Acid-base homeostasis is essential for life. The macromolecules upon which living organisms depend are sensitive to pH changes, and physiological systems use the equilibrium between carbon dioxide, bicarbonate, and protons to buffer their pH. Biological processes and environmental insults are constantly challenging an organism's pH; therefore, to maintain a consistent and proper pH, organisms need sensors that measure pH and that elicit appropriate responses. Mammals use multiple sensors for measuring both intracellular and extracellular pH, and although some mammalian pH sensors directly measure protons, it has recently become apparent that many pH-sensing systems measure pH via bicarbonate-sensing soluble adenylyl cyclase.

Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate.

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.

Soluble adenylyl cyclase is necessary and sufficient to overcome the block of axonal growth by myelin-associated factors.

Neurons in the CNS do not regenerate following injury; regeneration is blocked by inhibitory proteins in myelin, such as myelin-associated glycoprotein (MAG). Elevating neuronal levels of the second messenger cAMP overcomes this blocked axonal outgrowth. One way to elevate cAMP is pretreating neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF). However, pleiotropic effects and poor bioavailability make exogenous administration of neurotrophins in vivo problematic; therefore, alternative targets must be considered. In neurons, two families of adenylyl cyclases synthesize cAMP, transmembrane adenylyl cyclases (tmACs), and soluble adenylyl cyclase (sAC). Here, we demonstrate that sAC is the essential source of cAMP for BDNF to overcome MAG-dependent inhibition of neurite outgrowth. Elevating sAC in rat and mouse neurons is sufficient to induce neurite outgrowth on myelin in vitro and promotes regeneration in vivo. These results suggest that stimulators of sAC might represent a novel therapeutic strategy to promote axonal growth and regeneration.

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.

Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF.

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ß cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

cAMP and mitochondria.

Phosphorylation of mitochondrial proteins has emerged as a major regulatory mechanism for metabolic adaptation. cAMP signaling and PKA phosphorylation of mitochondrial proteins have just started to be investigated, and the presence of cAMP-generating enzymes and PKA inside mitochondria is still controversial. Here, we discuss the role of cAMP in regulating mitochondrial bioenergetics through protein phosphorylation and the evidence for soluble adenylyl cyclase as the source of cAMP inside mitochondria.

The uniqueness of on-demand male contraception.

Because nearly half of pregnancies worldwide are unintended, available contraceptive methods are inadequate. Moreover, due to the striking imbalance between contraceptive options available for men compared to the myriad of options available to women, there is an urgent need for new methods of contraception for men. This review summarizes ongoing efforts to develop male contraceptives highlighting the unique aspects particular to on-demand male contraception, where a man takes a contraceptive only when and as often as needed.

Pharmacological distinction between soluble and transmembrane adenylyl cyclases.

The second messenger cAMP is involved in a number of cellular signaling pathways. In mammals, cAMP is produced by either the hormonally responsive, G protein-regulated transmembrane adenylyl cyclases (tmACs) or by the bicarbonate- and calcium-regulated soluble adenylyl cyclase (sAC). To develop tools to differentiate tmAC and sAC signaling, we determined the specificity and potency of commercially available adenylyl cyclase inhibitors. In cellular systems, two inhibitors, KH7 and catechol estrogens, proved specific for sAC, and 2',5'-dideoxyadenosine proved specific for tmACs. These tools provide a means to define the specific contributions of the different families of adenylyl cyclases in cells and tissues, which will further our understanding of cell signaling.