Anti-inflammatory drug design by molecular hybridization approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Combined Study Using Ligand-Based Design, Synthesis, and Pharmacological Evaluation of Analogues of the Acetaminophen Ortho-Regioisomer with Potent Analgesic Activity

A ligand-based drug design study was performed to acetaminophen regioisomers as analgesic candidates employing quantum chemical calculations at the DFT/B3LYP level of theory and the 6-31G* basis set. To do so, many molecular descriptors were used such as highest occupied molecular orbital, ionization potential, HO bond dissociation energies, and spin densities, which might be related to quench reactivity of the tyrosyl radical to give N-acetyl-p-benzosemiquinone-imine through an initial electron withdrawing or hydrogen atom abstraction. Based on this in silico work, the most promising molecule, orthobenzamol, was synthesized and tested. The results expected from the theoretical prediction were confirmed in vivo using mouse models of nociception such as writhing, paw licking, and hot plate tests. All biological results suggested an antinociceptive activity mediated by opioid receptors. Furthermore, at 90 and 120 min, this new compound had an effect that was comparable to morphine, the standard drug for this test. Finally, the pharmacophore model is discussed according to the electronic properties derived from quantum chemistry calculations.

Structure-based drug design studies on a series of aldolase inhibitors

Human African trypanosomiasis, also known as sleeping sickness, is a major cause of death in Africa, and for which there are no safe and effective treatments available. The enzyme aldolase from Trypanosoma brucei is an attractive, validated target for drug development. A series of alkyl‑glycolamido and alkyl-monoglycolate derivatives was studied employing a combination of drug design approaches. Three-dimensional quantitative structure-activity relationships (3D QSAR) models were generated using the comparative molecular field analysis (CoMFA). Significant results were obtained for the best QSAR model (r2 = 0.95, non-cross-validated correlation coefficient, and q2 = 0.80, cross-validated correlation coefficient), indicating its predictive ability for untested compounds. The model was then used to predict values of the dependent variables (pKi) of an external test set,the predicted values were in good agreement with the experimental results. The integration of 3D QSAR, molecular docking and molecular dynamics simulations provided further insight into the structural basis for selective inhibition of the target enzyme.

Synthesis of Biologically Active Small Molecules: Different Approaches to Drug Design

In the past years, genome biology had disclosed an ever-growing kind of biological targets that emerged as ideal points for therapeutic intervention. Nevertheless, the number of new chemical entities (NCEs) translated into effective therapies employed in the clinic, still not observed. Innovative strategies in drug discovery combined with different approaches to drug design should be searched for bridge this gap. In this context organic synthetic chemistry had to provide for effective strategies to achieve biologically active small molecules to consider not only as potentially drug candidates, but also as chemical tools to dissect biological systems. In this scenario, during my PhD, inspired by the Biology-oriented Synthesis approach, a small library of hybrid molecules endowed with privileged scaffolds, able to block cell cycle and to induce apoptosis and cell differentiation, merged with natural-like cores were synthesized. A synthetic platform which joined a Domino Knoevenagel-Diels Alder reaction with a Suzuki coupling was performed in order to reach the hybrid compounds. These molecules can represent either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells. The biological profile expressed by some of these derivatives showed a well defined antiproliferative activity on leukemia Bcr-Abl expressing K562 cell lines. A parallel project regarded the rational design and synthesis of minimally structurally hERG blockers with the purpose of enhancing the SAR studies of a previously synthesized collection. A Target-Oriented Synthesis approach was applied. Combining conventional and microwave heating, the desired final compounds were achieved in good yields and reaction rates. The preliminary biological results of the compounds, showed a potent blocking activity. The obtained small set of hERG blockers, was able to gain more insight the minimal structural requirements for hERG liability, which is mandatory to investigate in order to reduce the risk of potential side effects of new drug candidates.

Conformationally homogeneous heterocyclic pseudotetrapeptides as three-dimensional scaffolds for rational drug design: receptor-selective somatostatin analogues

A would-be amide: A 1,4-disubstituted 1,2,3-triazole was used as a surrogate for a trans amide bond to create a library of 16 diastereomeric pseudotetrapeptides as beta-turn mimetics. High-resolution structural analysis indicated that these scaffolds adopt distinct, rigid, conformationally homogeneous beta-turn-like structures (see example), some of which bind somatostatin receptor subtypes selectively, and some of which show broad-spectrum activity.

The role of workplace absence as an organizational mechanism in predicting employee alcohol and drug disorders

Background. EAP programs for airline pilots in companies with a well developed recovery management program are known to reduce pilot absenteeism following treatment. Given the costs and safety consequences to society, it is important to identify pilots who may be experiencing an AOD disorder to get them into treatment. ^ Hypotheses. This study investigated the predictive power of workplace absenteeism in identifying alcohol or drug disorders (AOD). The first hypothesis was that higher absenteeism in a 12-month period is associated with higher risk that an employee is experiencing AOD. The second hypothesis was that AOD treatment would reduce subsequent absence rates and the costs of replacing pilots on missed flights. ^ Methods. A case control design using eight years (time period) of monthly archival absence data (53,000 pay records) was conducted with a sample of (N = 76) employees having an AOD diagnosis (cases) matched 1:4 with (N = 304) non-diagnosed employees (controls) of the same profession and company (male commercial airline pilots). Cases and controls were matched on the variables age, rank and date of hire. Absence rate was defined as sick time hours used over the sum of the minimum guarantee pay hours annualized using the months the pilot worked for the year. Conditional logistic regression was used to determine if absence predicts employees experiencing an AOD disorder, starting 3 years prior to the cases receiving the AOD diagnosis. A repeated measures ANOVA, t tests and rate ratios (with 95% confidence intervals) were conducted to determine differences between cases and controls in absence usage for 3 years pre and 5 years post treatment. Mean replacement costs were calculated for sick leave usage 3 years pre and 5 years post treatment to estimate the cost of sick leave from the perspective of the company. ^ Results. Sick leave, as measured by absence rate, predicted the risk of being diagnosed with an AOD disorder (OR 1.10, 95% CI = 1.06, 1.15) during the 12 months prior to receiving the diagnosis. Mean absence rates for diagnosed employees increased over the three years before treatment, particularly in the year before treatment, whereas the controls’ did not (three years, x = 6.80 vs. 5.52; two years, x = 7.81 vs. 6.30, and one year, x = 11.00cases vs. 5.51controls. In the first year post treatment compared to the year prior to treatment, rate ratios indicated a significant (60%) post treatment reduction in absence rates (OR = 0.40, CI = 0.28, 0.57). Absence rates for cases remained lower than controls for the first three years after completion of treatment. Upon discharge from the FAA and company’s three year AOD monitoring program, case’s absence rates increased slightly during the fourth year (controls, x = 0.09, SD = 0.14, cases, x = 0.12, SD = 0.21). However, the following year, their mean absence rates were again below those of the controls (controls, x = 0.08, SD = 0.12, cases, x¯ = 0.06, SD = 0.07). Significant reductions in costs associated with replacing pilots calling in sick, were found to be 60% less, between the year of diagnosis for the cases and the first year after returning to work. A reduction in replacement costs continued over the next two years for the treated employees. ^ Conclusions. This research demonstrates the potential for workplace absences as an active organizational surveillance mechanism to assist managers and supervisors in identifying employees who may be experiencing or at risk of experiencing an alcohol/drug disorder. Currently, many workplaces use only performance problems and ignore the employee’s absence record. A referral to an EAP or alcohol/drug evaluation based on the employee’s absence/sick leave record as incorporated into company policy can provide another useful indicator that may also carry less stigma, thus reducing barriers to seeking help. This research also confirms two conclusions heretofore based only on cross-sectional studies: (1) higher absence rates are associated with employees experiencing an AOD disorder; (2) treatment is associated with lower costs for replacing absent pilots. Due to the uniqueness of the employee population studied (commercial airline pilots) and the organizational documentation of absence, the generalizability of this study to other professions and occupations should be considered limited. ^ Transition to Practice. The odds ratios for the relationship between absence rates and an AOD diagnosis are precise; the OR for year of diagnosis indicates the likelihood of being diagnosed increases 10% for every hour change in sick leave taken. In practice, however, a pilot uses approximately 20 hours of sick leave for one trip, because the replacement will have to be paid the guaranteed minimum of 20 hour. Thus, the rate based on hourly changes is precise but not practical. ^ To provide the organization with practical recommendations the yearly mean absence rates were used. A pilot flies on average, 90 hours a month, 1080 annually. Cases used almost twice the mean rate of sick time the year prior to diagnosis (T-1) compared to controls (cases, x = .11, controls, x = .06). Cases are expected to use on average 119 hours annually (total annual hours*mean annual absence rate), while controls will use 60 hours. The cases’ 60 hours could translate to 3 trips of 20 hours each. Management could use a standard of 80 hours or more of sick time claimed in a year as the threshold for unacceptable absence, a 25% increase over the controls (a cost to the company of approximately of $4000). At the 80-hour mark, the Chief Pilot would be able to call the pilot in for a routine check as to the nature of the pilot’s excessive absence. This management action would be based on a company standard, rather than a behavioral or performance issue. Using absence data in this fashion would make it an active surveillance mechanism. ^

Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: Neuroprotective drug design

The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar–picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N-terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr-Leu-NH2 (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against β-amyloid toxicity in vitro. Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer’s disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Theranostic Fibers for Simultaneous Imaging and Drug Delivery

New methods for creating theranostic systems with simultaneous encapsulation of therapeutic, diagnostic, and targeting agents are much sought after. This work reports for the first time the use of coaxial electrospinning to prepare such systems in the form of core–shell fibers. Eudragit S100 was used to form the shell of the fibers, while the core comprised poly(ethylene oxide) loaded with the magnetic resonance contrast agent Gd(DTPA) (Gd(III) diethylenetriaminepentaacetate hydrate) and indomethacin as a model therapeutic agent. The fibers had linear cylindrical morphologies with clear core–shell structures, as demonstrated by electron microscopy. X-ray diffraction and differential scanning calorimetry proved that both indomethacin and Gd(DTPA) were present in the fibers in the amorphous physical form. This is thought to be a result of intermolecular interactions between the different components, the presence of which was suggested by infrared spectroscopy. In vitro dissolution tests indicated that the fibers could provide targeted release of the active ingredients through a combined mechanism of erosion and diffusion. The proton relaxivities for Gd(DTPA) released from the fibers into tris buffer increased (r1 = 4.79–9.75 s–1 mM–1; r2 = 7.98–14.22 s–1 mM–1) compared with fresh Gd(DTPA) (r1 = 4.13 s–1 mM–1 and r2 = 4.40 s–1 mM–1), which proved that electrospinning has not diminished the contrast properties of the complex. The new systems reported herein thus offer a new platform for delivering therapeutic and imaging agents simultaneously to the colon.

Characterizing bupropion metabolism, pharmacokinetics and drug-drug interaction liability

Thesis (Ph.D.)--University of Washington, 2016-06

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Integrating structural and input design of a 2-DOF high-speed parallel manipulator: A flexible model-based approach

This paper discusses the integrated design of parallel manipulators, which exhibit varying dynamics. This characteristic affects the machine stability and performance. The design methodology consists of four main steps: (i) the system modeling using flexible multibody technique, (ii) the synthesis of reduced-order models suitable for control design, (iii) the systematic flexible model-based input signal design, and (iv) the evaluation of some possible machine designs. The novelty in this methodology is to take structural flexibilities into consideration during the input signal design; therefore, enhancing the standard design process which mainly considers rigid bodies dynamics. The potential of the proposed strategy is exploited for the design evaluation of a two degree-of-freedom high-speed parallel manipulator. The results are experimentally validated. (C) 2010 Elsevier Ltd. All rights reserved.

Methadone maintenance and drug-related crime

Using data from an evaluation of methadone maintenance treatment, this study investigated factors associated with continued involvement irt crime during treatment, and in particular whether there appeared to be differences in effectiveness of treatment between different methadone clinics. The methodology was an observational study, in which 304 patients attending three low-intervention, private methadone clinics in Sydney were interviewed on three occasions over a twelve month period. Outcome measures were self-reported criminal activity and police department records of convictions. By self-report, crime dropped, promptly and substantially on entry to treatment, to a level of acquisitive crime about one-eighth that reported during the last addiction period. Analysis of official records indicated that rates of acquisitive convictions were significantly lower in the in-treatment period compared to prior to entry to treatment, corroborating the changes suggested by self-report. Persisting involvement in crime in treatment was predicted by two factors: the cost of persisting use of illicit drugs, particularly cannabis, and ASPD symptom count. Treatment factors also were independently predictive of continued involvement in crime. By both self-report and official records, and adjusting for subject factors, treatment at one clinic teas associated with greater involvement in crime. This clinic operated in a chaotic and poorly organized way. it is concluded that crime during methadone treatment is substantially lower than during street addiction, although the extent of reduction depends on the quality of treatment being delivered.