Jose A. Hernandez Prada

Structure-Based Identification of Small-Molecule Angiotensin-Converting Enzyme 2 Activators as Novel Antihypertensive Agents

Angiotensin-converting enzyme 2 (ACE2) is a key renin-angiotensin system enzyme involved in balancing the adverse effects of angiotensin II on the cardiovascular system, and its overexpression by gene transfer is beneficial in cardiovascular disease. Therefore, our objectives were 2-fold: to identify compounds that enhance ACE2 activity using a novel conformation-based rational drug discovery strategy and to evaluate whether such compounds reverse hypertension-induced pathophysiologies. We used a unique virtual screening approach. In vitro assays revealed 2 compounds (a xanthenone and resorcinolnaphthalein) that enhanced ACE2 activity in a dose-dependent manner. Acute in vivo administration of the xanthenone resulted in a dose-dependent transient and robust decrease in blood pressure (at 10 mg/kg, spontaneously hypertensive rats decreased 71±9 mm Hg and Wistar-Kyoto rats decreased 21±8 mm Hg; P<0.05). Chronic infusion of the xanthenone (120 μg/day) resulted in a modest decrease in the spontaneously hypertensive rat blood pressure (17 mm Hg; 2-way ANOVA; P<0.05), whereas it had no effect in Wistar-Kyoto rats. Strikingly, the decrease in blood pressure was also associated with improvements in cardiac function and reversal of myocardial, perivascular, and renal fibrosis in the spontaneously hypertensive rats. We conclude that structure-based screening can help identify compounds that activate ACE2, decrease blood pressure, and reverse tissue remodeling. Administration of ACE2 activators may be a valid strategy for antihypertensive therapy.

Discovery of Novel DNA Gyrase Inhibitors by High-Throughput Virtual Screening

ABSTRACT The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region–containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 Å) and a pair of V-type domains (to 1.85 Å) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.

A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition.

Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.

Structure-Based Discovery of a Novel Angiotensin-Converting Enzyme 2 Inhibitor

Angiotensin-converting enzyme 2 (ACE2) is considered an important therapeutic target for controlling cardiovascular diseases and severe acute respiratory syndrome (SARS) outbreaks. Recently solved high-resolution crystal structures of the apo-bound and inhibitor-bound forms of ACE2 have provided the basis for a novel molecular docking approach in an attempt to identify ACE2 inhibitors and compounds that block SARS coronavirus spike protein-mediated cell fusion. In this study, ≈140 000 small molecules were screened by in silico molecular docking. In this structure–activity relation study, the molecules with the highest predicted binding scores were identified and assayed for ACE2 enzymatic inhibitory activity and for their ability to inhibit SARS coronavirus spike protein-mediated cell fusion. This approach identified N-(2-aminoethyl)-1 aziridine-ethanamine as a novel ACE2 inhibitor that also is effective in blocking the SARS coronavirus spike protein-mediated cell fusion. Thus, the molecular docking approach resulting in the inhibitory capacity of N-(2-aminoethyl)-1 aziridine-ethanamine provides an attractive small molecule lead compound on which the development of more effective therapeutic agents could be developed to modulate hypertension and for controlling SARS infections.

Proton transfer in a Thr200His mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc‐bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A‐T200H HCAII), effectively relocating the proton shuttle residue ∼ 2 Å closer to the zinc‐bound hydroxide compared to wild type HCA II. The crystal structure of H64A‐T200H HCA II at 1.8 Å resolution shows the side chain of His200 directly hydrogen‐bonded with the zinc‐bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration‐dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A‐T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc‐bound hydroxide, in analogy with proton transfer involving His64 in wild‐type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc‐bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen‐bonded to the zinc‐bound hydroxide, can adopt the correct distance geometry to support proton transfer. Proteins 2005.

Crystallization and preliminary X-ray analysis of VCBP3 from Branchiostoma floridae.

VCBPs represent a family of proteins with highly diversified immunoglobulin-like variable regions in species thought to lack an adaptive immune system. These proteins are expected to reveal important structural and functional features that could be highly informative in projecting the evolutionary history of the adaptive immune response. Preliminary X-ray diffraction data from amphioxus (Branchiostoma floridae) VCBP3 crystals were collected to 2.4 A resolution and reduced to primitive trigonal space groups P3(1(2))21. Unit-cell parameters are a = b = 58.99, c = 79.21 A, alpha = beta = 90, gamma = 120 degrees . Two distinct crystallization conditions yielded crystals with similar morphologies and these crystals are isomorphous to each other.

Molecular Modeling Optimization of Anticoagulant Pyridine Derivatives

Intravascular clotting remains a major health problem in the United States, the most prominent being deep vein thrombosis, pulmonary embolism and thromboembolic stroke. Previous reports on the use of pyridine derivatives in cardiovascular drug development encourage us to pursue new types of compounds based on a pyridine scaffold. Eleven pyridine derivatives (oximes, semicarbazones, N-oxides) previously synthesized in our laboratories were tested as anticoagulants on pooled normal plasma using the prothrombin time (PT) protocol. The best anticoagulant within the oxime series was compound AF4, within the oxime N-oxide series was compound AF4-N-oxide, and within the semicarbazone series, compound MD1-30Y. We also used a molecular modeling approach to guide our efforts, and found that there was good correlation between coagulation data and computational energy scores. Molecular docking was performed to target the active site of thrombin with the DOCK v5.2 package. The results of molecular modeling indicate that improvement in anticoagulant activities can be expected by functionalization at the three-position of the pyridine ring and by N-oxide formation. Results reported here prove the suitability of DOCK in the lead optimization process.

Crystallization and X-ray diffraction analysis of a novel immune-type receptor from Ictalurus punctatus and phasing by selenium anomalous dispersion methods.

X-ray diffraction data from crystals of a novel immune-type receptor (NITR10 from the catfish Ictalurus punctatus) were collected to 1.65 A resolution and reduced to the primitive hexagonal lattice. Native and selenomethionine derivatives of NITR10 crystallized under different conditions yielded P3(1)21 crystals. SeMet NITR10 was phased to a correlation coefficient of 0.77 by SAD methods and experimental electron-density maps were calculated to 1.65 A. Five NITR10 molecules are predicted to be present in the asymmetric unit based on the Matthews coefficient.

Cardiovascular protection by angiotensin- converting enzyme 2: a new paradigm

A novel angiotensin-converting enzyme (ACE) homolog, named ACE2, was recently described. ACE2 degrades Ang II, a peptide with vasoconstrictive and proliferative effects, to generate Ang-(1-7), which, acting through its receptor Mas, exerts vasodilatory and antiproliferative actions. In addition, ACE2 is a multifunctional enzyme and its actions on other vasoactive peptides can also contribute to its vasoactive effects. The discovery of ACE2 corroborates the establishment of two counter-regulatory arms within the renin-angiotensin system. The first arm is formed by the classical pathway involving the ACE-Ang II-AT(1)-receptor axis, and the second arm is constituted by the ACE2-Ang-(1-7)-Mas-receptor axis. Owing to its characteristics, the ACE2-Ang-(1-7)-Mas axis may represent new possibilities for developing novel therapeutic strategies for the treatment of hypertension and cardiovascular diseases. In this review, we will summarize the biochemical and pathophysiological aspects of ACE2 with particular focus on its role in the heart.