Carbonic Anhydrase Inhibitor Modulation of Intraocular Pressure Is Independent of Soluble Adenylyl Cyclase.

Purpose: We investigated whether a clinically used carbonic anhydrase inhibitor (CAIs) can modulate intraocular pressure (IOP) through soluble adenylyl cyclase (sAC) signaling. Methods: IOP was measured 1 h after topical treatment with brinzolamide, a topically applied and clinically used CAIs, using direct cannulation of the anterior chamber in sAC knockout (KO) mice or C57BL/6J mice in the presence or absence of the sAC inhibitor (TDI-10229). Results: Mice treated with the sAC inhibitor TDI-10229 had elevated IOP. CAIs treatment significantly decreased increased intraocular pressure (IOP) in wild-type, sAC KO mice, as well as TDI-10229-treated mice. Conclusions: Inhibiting carbonic anhydrase reduces IOP independently from sAC in mice. Our studies suggest that the signaling cascade by which brinzolamide regulates IOP does not involve sAC.

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.

Corrigendum: Assessing potency and binding kinetics of soluble adenylyl cyclase (sAC) inhibitors to maximize therapeutic potential.

[This corrects the article DOI: 10.3389/fphys.2022.1013845.].

On-demand male contraception via acute inhibition of soluble adenylyl cyclase.

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates.

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Assessing potency and binding kinetics of soluble adenylyl cyclase (sAC) inhibitors to maximize therapeutic potential.

In mammalian cells, 10 different adenylyl cyclases produce the ubiquitous second messenger, cyclic adenosine monophosphate (cAMP). Amongst these cAMP-generating enzymes, bicarbonate (HCO3 -)-regulated soluble adenylyl cyclase (sAC; ADCY10) is uniquely essential in sperm for reproduction. For this reason, sAC has been proposed as a potential therapeutic target for non-hormonal contraceptives for men. Here, we describe key sAC-focused in vitro assays to identify and characterize sAC inhibitors for therapeutic use. The affinity and binding kinetics of an inhibitor can greatly influence in vivo efficacy, therefore, we developed improved assays for assessing these efficacy defining features.

Strategies to safely target widely expressed soluble adenylyl cyclase for contraception.

In humans, the prototypical second messenger cyclic AMP is produced by 10 adenylyl cyclase isoforms, which are divided into two classes. Nine isoforms are G protein coupled transmembrane adenylyl cyclases (tmACs; ADCY1-9) and the 10th is the bicarbonate regulated soluble adenylyl cyclase (sAC; ADCY10). This review details why sAC is uniquely druggable and outlines ways to target sAC for novel forms of male and female contraception.

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.

Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

MRSA Isolates from United States Hospitals Carry dfrG and dfrK Resistance Genes and Succumb to Propargyl-Linked Antifolates.

Antibiotic resistance is a rapidly evolving health concern that requires a sustained effort to understand mechanisms of resistance and to develop new agents that overcome those mechanisms. The dihydrofolate reductase (DHFR) inhibitor, trimethoprim (TMP), remains one of the most important orally administered antibiotics. However, resistance through chromosomal mutations and mobile, plasmid-encoded insensitive DHFRs threatens the continued use of this agent. We are pursuing the development of new propargyl-linked antifolate (PLA) DHFR inhibitors designed to evade these mechanisms. While analyzing contemporary TMP-resistant clinical isolates of methicillin-resistant and sensitive Staphylococcus aureus, we discovered two mobile resistance elements, dfrG and dfrK. This is the first identification of these resistance mechanisms in the United States. These resistant organisms were isolated from a variety of infection sites, show clonal diversity, and each contain distinct resistance genotypes for common antibiotics. Several PLAs showed significant activity against these resistant strains by direct inhibition of the TMP resistance elements.

Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations.

Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme.

Utilizaçäo da água potável como veículo de nutrientes: estudos experimentais com ferro / Use of drinking water as a vehicle of nutrients: experimental studies with iron

Esta investigaçäo é parte de um projeto mayor com o objetivo de estudar a possibilidade de se usar água potável como carreador de nutrientes, tais como o ferro, para uso de comunidades. Para verificar tal possibilidade vários sais de ferro em diferentes concentraçöes foram adicionados água e analisados quanto a seus efeitos em relaçäo à cor e à turbidez. Os resultados destes estudos mostraram que o citrato férrico amoniacal foi o melhor sal no que diz respeito à manutençäo das características físicas de água potável clorada. Também foi estudada a biodisponibilidade de diversos sais de ferro adicionados a água para prevenir a anemia em ratos. Os ensaios biológicos mostraron que as formas mais adequadas de sais de ferro em água para prevenir foram: citrato férrico amoniacal, sulfato ferroso e gluconato de ferro

Dosagem de aflatoxina B1 em amendoim e doces de amendoim / Aflatoxins B1 dosage in peanut and peanut candies

Aflatoxinas säo do grupo metabolitos cancerígenos altamente tóxicos produzidos por fungos do tipo Aspergillus flavus. O desenvolvimento desses fungos no amendoim está relacionado com o tempo, temperatura e umidade do local em que este produto é estocado. a legislaçäo brasileira (Resoluçäo 34/76 da CNNPA) tolera um limite de 30 ppb (microng/Kg), daí a importância do seu controle. A dosagem de aflatoxinas foi feita por cromatografia em camada delgada em amendoim e em doces de amendoim procedentes da regiäo de Ribeiräo Preto, SP., de janeiro de 1983 a dezembro de 1985. Foi encontrada aflatoxina B1 em 67,2% das amostras analisadas: acima de 30 ppb foi detectada em 24,5% e abaixo em 32,7%. No pé-de-moleque, foi detectada aflatoxina B1 em 54,6% das amostras com 18,2% acima e 36,4% abaixo de 30 ppb. Na paçoca, em 66,6% das amostras, com 13,3% acima e 53,3% abaixo de 30 ppb. Na bala amendoim, näo foi detectada aflatoxina B1 acima de 30 ppb, e, apenas em 12,5% das amostras, em quantidades inferior a 30 ppb