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Tofacitinib is an oral, small molecule Janus kinase inhibitor for the treatment of ulcerative colitis (UC). We report a model-informed drug development approach for bridging efficacy from immediate-release (IR) to extended-release (XR) tofacitinib formulations in patients with UC. IR-XR efficacy bridging was supported by exposure-response analysis of phase 3 induction/maintenance studies of the IR formulation in UC to identify exposure metrics relevant for efficacy. Pharmacokinetic studies in healthy subjects were used to confirm similarity of relevant exposure metrics of tofacitinib IR 5 mg twice daily to XR 11 mg once daily, and tofacitinib IR 10 mg twice daily to XR 22 mg once daily, thereby bridging efficacy between IR and XR formulations. Food effect was evaluated at both XR formulation dose levels. Exposure-response analysis demonstrated that area under the plasma concentration-time curve (average plasma concentration) was a relevant predictor of efficacy. Pharmacokinetic studies demonstrated that area under the plasma concentration-time curve was equivalent between formulations under single-dose and steady-state conditions, and other exposure metrics were also similar. These results also supported bridging of safety data for IR-XR formulations. Food had no impact on tofacitinib XR exposure. These data support efficacy/safety bridging of IR-XR formulations in patients with UC.

Abrocitinib is a Janus kinase 1-selective inhibitor for the treatment of moderate-to-severe atopic dermatitis. Abrocitinib is eliminated primarily by metabolism involving cytochrome P450 (CYP) enzymes. Abrocitinib pharmacologic activity is attributable to the unbound concentrations of the parent molecule and 2 active metabolites, which are substrates of organic anion transporter 3 (OAT3). The sum of potency-adjusted unbound exposures of abrocitinib and its 2 active metabolites is termed the abrocitinib active moiety. We evaluated effects of CYP inhibition, CYP induction, and OAT3 inhibition on the pharmacokinetics of abrocitinib, its metabolites, and active moiety.Three fixed-sequence, open-label, phase I studies in healthy adult volunteers examined the drug-drug interactions (DDIs) of oral abrocitinib with fluvoxamine and fluconazole, rifampin, and probenecid.Co-administration of abrocitinib with fluvoxamine or fluconazole increased the area under the plasma concentration-time curve from time 0 to infinity (AUCIt is important to consider the effects of DDIs on the abrocitinib active moiety when making dosing recommendations. Co-administration of strong CYP2C19/2C9 inhibitors or CYP inducers impacted exposure to the abrocitinib active moiety. A dose reduction by half is recommended if abrocitinib is co-administered with strong CYP2C19 inhibitors, whereas co-administration with strong CYP2C19/2C9 inducers is not recommended. No dose adjustment is required when abrocitinib is administered with OAT3 inhibitors.NCT03634345, NCT03637790, NCT03937258.

Abrocitinib is a selective Janus kinase 1 inhibitor for the treatment of moderate to severe atopic dermatitis (AD). To assess the relationship between abrocitinib plasma concentrations and heart rate (HR)-corrected QT (QTc) and HR and calculate the effect of abrocitinib on these parameters at supratherapeutic concentrations, 36 healthy volunteers received single doses of abrocitinib 600 mg, placebo, and moxifloxacin 400 mg in a 3-period crossover study. The relationship between change from baseline in Fridericia-corrected QTc (∆QTcF) values and abrocitinib plasma concentrations was modeled using a prespecified linear mixed-effects model. The 90%CIs for time-matched placebo-corrected ∆QTcF (∆∆QTcF) were calculated from model parameter estimates and assessed against the regulatory threshold (10 millisecond) at the predicted supratherapeutic concentration in patients with atopic dermatitis (2156 ng/mL). Mean (90%CI) time-matched placebo-corrected change from baseline in HR (∆∆HR) was calculated similarly. At the supratherapeutic concentration, mean (90%CI) estimates for ∆∆QTcF and ∆∆HR were 6.00 (4.52-7.49) milliseconds and 6.51 (5.23-7.80) bpm, respectively. Despite a concentration-dependent effect on ∆QTcF and ∆HR, with statistically significant slopes (90%CI) of 0.0026 (0.0018-0.0035) milliseconds/(ng/mL) and 0.0031 (0.0024-0.0038) bpm/(ng/mL), respectively, abrocitinib does not have a clinically significant effect on QTc interval or HR at supratherapeutic exposures.

(1)H NMR and isothermal titration calorimetry (ITC) experiments were employed to obtain reliable thermodynamic data for the formation of the 1:1 inclusion complexes of fullerenes C(60) and C(70) with the buckycatcher (C(60)H(28)). NMR measurements were done in toluene-d8 and chlorobenzene-d5 at 288, 298, and 308 K, while the ITC titrations were performed in toluene, chlorobenzene, o-dichlorobenzene, anisole, and 1,1,2,2-tetrachloroethane at temperatures from 278 to 323 K. The association constants, K(a), obtained with both techniques are in very good agreement. The thermodynamic data obtained by ITC indicate that generally the host-guest association is enthalpy-driven. Interestingly, the entropy contributions are, with rare exceptions, slightly stabilizing or close to zero. Neither ΔH nor ΔS is constant over the temperature range studied, and these thermodynamic functions exhibit classical enthalpy/entropy compensation. The ΔCp values calculated from the temperature dependence of the calorimetric ΔH values are negative for the association of both fullerenes with the buckycatcher in toluene. The negative ΔCp values are consistent with some desolvation of the host-cavity and the guest in the inclusion complexes, C(60)@C(60)H(28) and C(70)@C(60)H(28).

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1-G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na(+) and K(+) G-quadruplex-UP1 complexes (ΔH values of -43 and -19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na(+) form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10(8) M(-1) (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10(6) M(-1) (strand)]. Circular dichroism spectroscopy reveals the Na(+) form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.

G-quadruplexes have shown great promise as chemotherapeutic targets, probably by inhibiting telomere elongation or downregulating oncogene expression. There have been many G-quadruplex ligands developed over the years but only a few have drug-like properties. Consequently only a few G-quadruplex ligands have entered clinical trials as cancer chemotherapeutic agents. The DNA minor groove ligand, berenil (diminazene aceturate or DMZ), is used to treat animal trypanosomiasis and hence its toxicological profile is already known, making it an ideal platform to engineer into new therapeutics. Herein, using a plethora of biophysical methods including UV, NMR, MS and ITC, we show that DMZ binds to several G-quadruplexes with a Kd of ∼1 nM. This is one of the strongest G-quadruplex binding affinities reported to date and is 10(3) tighter than the berenil affinity for an AT-rich duplex DNA. Structure-activity-relationship studies demonstrate that the two amidine groups on DMZ are important for binding to both G-quadruplex and duplex DNA. This work reveals that DMZ or berenil is not as selective for AT-rich duplexes as originally thought and that some of its biological effects could be manifested through G-quadruplex binding. The DMZ scaffold represents a good starting point to develop new G-quadruplex ligands for cancer cell targeting.

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Isothermal titration calorimetry (ITC) is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g., K(eq) (or ΔG), ΔH, ΔS, and n) for a ligand-binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combining equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example, one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models, for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding, need to be developed on a case-by-case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the nonlinear regression analysis of a multiple-binding-site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g., up to nine parameters in the three-binding-site model) yields thermodynamic parameters with acceptable accuracy.

Isothermal titration calorimetry (ITC) can be used to study the thermodynamics of enzyme substrate binding or the kinetics of substrate turnover (or both). Substrate-binding interactions are observed in a typical ITC titration experiment in which the heat change for the addition of an aliquot of substrate to a solution containing the enzyme is determined for a number of titrant (i.e., substrate) injections and the data fit for the thermodynamic parameters (ΔG, ΔH, and -TΔS) for substrate binding. Of course, these measurements must be made under conditions where the substrate binds but does not turnover. In the ITC "kinetics" experiment, the power change observed after injection of an excess of substrate into a solution of the enzyme is a direct measure of the rate at which substrate is converted to product, and the ITC data can be analyzed for the kinetic parameters (Vmax, kcat, KM, and kcat/KM). The ITC technique is particularly versatile in that it can be applied to systems where there might not be a change in a spectroscopic signal for either substrate binding or the reaction of the substrate to form product. A complication is that if there are competing reactions, for example, buffer protonation, or product binding, to name just two, the enthalpy change measured for either substrate binding or for substrate turnover will be a summation of all of the reaction heats. Enzyme studies are typically done in buffered solutions at constant pH. The general, and often incorrect, assumption is that the buffer components are simply spectators and not participants in either substrate binding or substrate turnover. This chapter describes how we have used ITC measurements to identify problem buffers that impact the kinetics for an enzyme catalyzed reaction. Herein, we show the effects of several buffers on the steady-state kinetics for the conversion of the substrate, 3,4-dihydroxyphenyl acetate (homoprotocatechuate), to the ring-opened product, 5-carboxymethyl-2-hydroxymuconic semialdehyde by the nonheme iron(II) metalloenzyme, homoprotocatechuate 2,3-dioxygenase. Several buffers were observed to engage in buffer/enzyme interactions within the active site pocket. These enzyme-buffer interactions were shown to inhibit substrate turnover and to contribute additional enthalpy terms to the overall heat of reaction observed for substrate turnover (and for substrate binding).

Isothermal titration calorimetry (ITC) can be used to study the thermodynamics of enzyme substrate binding or the kinetics of substrate turnover (or both). Substrate-binding interactions are observed in a typical ITC titration experiment in which the heat change for the addition of an aliquot of substrate to a solution containing the enzyme is determined for a number of titrant (i.e., substrate) injections and the data fit for the thermodynamic parameters (ΔG, ΔH, and -TΔS) for substrate binding. Of course, these measurements must be made under conditions where the substrate binds but does not turnover. In the ITC "kinetics" experiment, the power change observed after injection of an excess of substrate into a solution of the enzyme is a direct measure of the rate at which substrate is converted to product, and the ITC data can be analyzed for the kinetic parameters (Vmax, kcat, KM, and kcat/KM). The ITC technique is particularly versatile in that it can be applied to systems where there might not be a change in a spectroscopic signal for either substrate binding or the reaction of the substrate to form product. A complication is that if there are competing reactions, for example, buffer protonation, or product binding, to name just two, the enthalpy change measured for either substrate binding or for substrate turnover will be a summation of all of the reaction heats. Enzyme studies are typically done in buffered solutions at constant pH. The general, and often incorrect, assumption is that the buffer components are simply spectators and not participants in either substrate binding or substrate turnover. This chapter describes how we have used ITC measurements to identify problem buffers that impact the kinetics for an enzyme catalyzed reaction. Herein, we show the effects of several buffers on the steady-state kinetics for the conversion of the substrate, 3,4-dihydroxyphenyl acetate (homoprotocatechuate), to the ring-opened product, 5-carboxymethyl-2-hydroxymuconic semialdehyde by the nonheme iron(II) metalloenzyme, homoprotocatechuate 2,3-dioxygenase. Several buffers were observed to engage in buffer/enzyme interactions within the active site pocket. These enzyme-buffer interactions were shown to inhibit substrate turnover and to contribute additional enthalpy terms to the overall heat of reaction observed for substrate turnover (and for substrate binding).

Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K = 1.1 ± 0.6 × 10(6)) and enthalpy change (ΔH = -17.0 ± 1.7 kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.

The protein and gold nanoparticle (AuNP) interfacial interaction has broad implications for biological and biomedical applications of AuNPs. In situ characterization of the morphology and structural evolution of protein on AuNPs is difficult. We have found that the protein coating layer formed by bovine serum albumin (BSA) on AuNP is highly permeable to further organothiol adsorption. Using mercaptobenzimidazole (MBI) as a molecular probe, it is found that BSA interaction with AuNP is an exceedingly lengthy process. Structural modification of BSA coating layer on AuNP continues even after 2 days’ aging of the (AuNP/BSA) mixture. While BSA is in a near full monolayer packing on the AuNPs, it passivates only up to 30% of the AuNP surfaces against MBI adsorption. Aging reduces the kinetics of the MBI adsorption. However, even in the most aged BSA-coated AuNP (3 days), 80% of the MBI adsorption occurs within the first 5 min of the MBI addition to the (AuNP/BSA) mixture. The possibility of MBI displacing the a...

Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect.

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P1 promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K+] and 130 mM [K+]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).

Background The human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure. Methods Isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na+] or [K+] ≈ 0.15 M). Results The apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 108 to 1010 M− 1 with the two lower-affinity sites exhibiting a K in the range of 104 to 105 M− 1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 104 to 105 M− 1. Conclusions In the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands. General significance The preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Study design This is a cadaveric biomechanical study evaluating the biomechanical properties of a novel spinopelvic fixation technique with percutaneous lumbo-sacro-iliac (LSI) screws in an unstable total sacrectomy model. Objective To compare standard posterior dual rod spinopelvic fixation alone with dual rod fixation supplemented with LSI screw fixation. Summary of background data Primary or metastatic tumors of the sacrum requiring a total sacrectomy can result in spinopelvic instability if inadequate fixation is achieved. Many fixation techniques have been proposed to address this instability. However, to date, an optimal fixation technique has not been established. Materials and methods Ten fresh-frozen cadaveric spinopelvic specimens were randomized according to bone mineral density (BMD) to either posterior rod fixation (control group) or posterior rod fixation with supplemental LSI screws (LSI group). After fixation, a total sacrectomy of each specimen was performed. Specimens where then potted and axially loaded in a caudal direction. Stiffness, yield load, energy absorbed at yield load, ultimate load, and energy absorbed at ultimate load were computed. A Student t test was used for statistical analysis with significance set at P Results The average age and BMD were not significantly different between the control and LSI groups (age: P=0.255; BMD: P=0.810). After normalizing for BMD, there were no significant differences detected for any of the biomechanical parameters measured between the 2 fixation techniques: stiffness (P=0.857), yield load (P=0.219), energy at yield load (P=0.293), ultimate load (P=0.407), and energy at ultimate load (P=0.773). However, both fixation techniques were able to withstand physiological loads. Conclusions Our study did not demonstrate any biomechanical advantage for supplemental LSI screw fixation in our axial loading model. However, given the theoretical advantage of this percutaneous technique, further studies are warranted that take into account forward bending and sagittal stability.

Dissipative optomechanics has some advantages in cooling compared to the conventional dispersion dominated systems. Here, we study the optical response of a cantilever-like, silica, microsphere pendulum, evanescently coupled to a fiber taper. In a whispering gallery mode resonator the cavity mode and motion of the pendulum result in both dispersive and dissipative optomechanical interactions. This unique mechanism leads to an experimentally observable, asymmetric response function of the transduction spectrum which can be explained using coupled-mode theory. The optomechanical transduction, and its relationship to the external coupling gap, are investigated and we show that the experimental behavior is in good agreement with the theoretical predictions. A deep understanding of this mechanism is necessary to explore trapping and cooling in dissipative optomechanical systems., 5 pages

The tunability of an optical cavity is an essential requirement for many areas of research. Here, we use the Pound-Drever-Hall technique to lock a laser to a whispering gallery mode (WGM) of a microbubble resonator, to show that linear tuning of the WGM, and the corresponding locked laser, display almost zero hysteresis. By applying aerostatic pressure to the interior surface of the microbubble resonator, optical mode shift rates of around $58$ GHz/MPa are achieved. The microbubble can measure pressure with a detection limit of $2\times 10^{-4}$ MPa, which is an improvement made on pressure sensing using this device. The long-term frequency stability of this tuning method for different input pressures is measured. The frequency noise of the WGM measured over $10$ minutes for an input pressure of $0.5$ MPa, has a maximum standard deviation of $36$ MHz., Comment: 5 pages, 4 figures

Before radiography was invented, spondylolisthesis was commonly mistaken for other prevalent diseases occurring at that time such as Pott’s disease. Since then, our knowledge of the condition has tremendously improved and that has helped with the search for its etiology, diagnosis, and treatment. The treatment of spondylolisthesis used to consist solely of nonoperative management with various types and combinations of casting and bedrest. However, since the introduction of surgical fusion techniques, there are various ways to treat patients who have failed conservative treatment. The general principles usually involve posterior, anterior, or combined fusion with or without instrumentation, and decompression if neurologic deficits are encountered. Reduction can be obtained with instrumentation, but complications are high. Although these concepts can be approached through multiple surgical approaches, the trend has been to combine them into a one-stage approach to minimize morbidity.

While the antitumor activity of P(4+) is relatively well understood, the binding mechanism and thermodynamics for formation of (P(4+)·DNA) complexes remain in question. The thermodynamic parameters (Ka, ΔG, ΔH, and -TΔS) for formation of DNA complexes of the ruthenium dimer, [(phen)2Ru(tatpp)Ru(phen)2](4+) (abbreviated as P(4+)), where phen is 1,10-phenanthroline and tatpp is 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3″,2″-1:2‴,3‴-n]-pentacene, were determined using isothermal titration calorimetry. Calorimetric and spectroscopic titration experiments were performed in which P(4+) was added to three duplex DNAs of different lengths. We determined that P(4+) binds to duplex DNA at 298 K with modest affinity (Ka ≈ 3.8 × 10(5) M(-1), ΔG ≈ -7.6 kcal/mol), that the enthalpy change is unfavorable (ΔH ≈ +2.1 kcal/mol), and that complex formation is driven by a large favorable change in entropy (-TΔS ≈ -9.7 kcal/mol). These thermodynamic values were found to be approximately independent of the length of the DNA, and the stoichiometry of the (P(4+)·DNA) complexes was determined to be 1 P(4+)/2 DNA bp, at least for the two shorter DNAs. On the basis of the thermodynamic parameters, and the binding stoichiometry (verified in ESI-MS experiments), we conclude that P(4+) is intercalating between two adjacent DNA base pairs and that the neighbor sites on either side of the bound ligand are excluded from binding additional P(4+).

Objective.—To evaluate the analgesic efficacy and safety of a single 20- or 40-mg dose of valdecoxib compared with placebo in treatment of a single, acute, moderate or severe migraine headache, with or without aura. Background.—Valdecoxib, an oral COX-2 specific inhibitor, is indicated for relief of the signs and symptoms of rheumatoid arthritis and osteoarthritis and treatment of primary dysmenorrhea. This study assessed the optimal dose of valdecoxib for treatment of a single, acute, moderate to severe migraine headache. Methods.—This was a double-blind, randomized, placebo- and active-controlled, multicenter, single-dose (primary end point) and multiple-dose (secondary end point), 56-day study of valdecoxib in the treatment of a single, acute, moderate or severe migraine headache, with or without aura. Migraine headaches were diagnosed according to International Headache Society (IHS) criteria. The primary efficacy end point was headache response (defined as reduction of headache pain intensity from moderate or severe to mild or none) at 2 hours postdose. Patients assessed their headache pain intensity and presence or absence of migraine-associated nausea, vomiting, phonophobia, and photophobia at intervals from 0 to 24 hours postdose. Sumatriptan 50 mg (encapsulated, in standard method, to maintain blinding) was included as a positive control for assay sensitivity. No statistical comparisons were performed between active treatment arms (valdecoxib 20 mg, valdecoxib 40 mg, and sumatriptan 50 mg). Adverse events and safety parameters were monitored throughout the study. Results.—In the intent-to-treat population of 570 patients (135 valdecoxib 20 mg, 151 valdecoxib 40 mg, 143 sumatriptan, and 141 placebo), no significant differences in baseline demographics among treatment groups were observed. The headache response rate with valdecoxib 40 mg and sumatriptan 50 mg was significantly greater than that with placebo at all time points from 2 to 24 hours postdose. With valdecoxib 20 mg, headache response rate was significantly greater than placebo from 2 to 4 hours. Significantly fewer patients treated with valdecoxib 40 mg, compared with placebo, experienced nausea, vomiting, and phonophobia at 2 hours postdose. Conclusions.—A single 40-mg dose of valdecoxib is effective and well tolerated in treatment of migraine headache pain and associated symptoms.

We review the method of producing adiabatic optical micro- and nanofibers using a hydrogen/oxygen flame brushing technique. The flame is scanned along the fiber, which is being simultaneously stretched by two translation stages. The tapered fiber fabrication is reproducible and yields highly adiabatic tapers with either exponential or linear profiles. Details regarding the setup of the flame brushing rig and the various parameters used are presented. Information available from the literature is compiled and further details that are necessary to have a functioning pulling rig are included. This should enable the reader to fabricate various taper profiles, while achieving adiabatic transmission of ∼99% for fundamental mode propagation. Using this rig, transmissions ranging from 85% to 95% for higher order modes in an optical nanofiber have been obtained.

The potential applications of protein-functionalized gold nanoparticles (AuNPs) have motivated many studies characterizing protein-AuNP interactions. However, the lack of detailed structural information has hindered our ability to understand the mechanism of protein adsorption on AuNPs. In order to determine the structural perturbations that occur during adsorption, hydrogen/deuterium exchange (HDX) of amide protons was measured for two proteins by NMR. Specifically, we measured both slow (5-300 min) and fast (10-500 ms) H/D exchange rates for GB3 and ubiquitin, two well-characterized proteins. Overall, amide exchange rates are very similar in the presence and absence of AuNPs, supporting a model where the adsorbed protein remains largely folded on the AuNP surface. Small differences in exchange rates are observed for several loop residues, suggesting that the secondary structure remains relatively rigid while loops and surface residues can experience perturbations upon binding. Strikingly, several of these residues are close to lysines, which supports a model where positive surface residues may interact favorably with AuNP-bound citrate. Because these proteins appear to remain folded on AuNP surfaces, these studies suggest that it may be possible to engineer functional AuNP-based nanoconjugates without the use of chemical linkers.

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II β-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

The formation of two different minor groove complexes between netropsin and A2T2 DNA has been attributed to specific binding and hydration effects. In this study, we have examined the effect of added osmolyte (e.g., TEG or betaine) on the binding of netropsin to a hairpin DNA, d(CGCGAATTCGCGTC-TCCGCGAATTCGCG)-3, having a single A2T2 binding site. Netropsin binding to this DNA construct is described by a two fractional site model with a saturation stoichiometry of 1:1. Free energy changes, ΔGi, for formation of both complex I and complex II decrease continuously as osmolyte is added (e.g., ΔG1 decreases by 1.3 kcal/mol and ΔG2 decreases by 0.8 kcal/mol in 4 m osmolyte vs buffer). The negative ΔCp values for formation of both complexes, I and II, are largely unaffected by the addition of osmolyte. Formation of complex I is accompanied by the acquisition of 31 water molecules vs 19 waters for complex II. The most significant difference between the two osmolytes is that betaine diminishes the fractional formation of the complex II species, virtually eliminating complex II at 2 m. Addition of osmolyte or a decrease in the temperature have approximately the same effect on DNA hydration and on the thermodynamics of netropsin binding.

We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5–5.5). In terms of drug targeting, the question is whether c-MYC promoter sequence i-motifs will exist in the nucleus at neutral pH. In this work, we have investigated the stability of a mutant c-MYC i-motif in solutions containing a molecular crowding agent. The crowded nuclear environment was modeled by the addition of up to 40% w/w polyethylene glycols having molecular weights up to 12,000 g/mol. CD and DSC were used to establish the presence and stability of c-MYC i-motifs in buffer solutions over the pH range 4 to 7. We have shown that the c-MYC i-motif can exist as a stable structure at pH values as high as 6.7 in crowded solutions. Generic dielectric constant effects, e.g., a shift in the pKa of cytosine by more than 2 units (e.g., 4.8 to 7.0), or the formation of non-specific PEG/DNA complexes appear to contribute insignificantly to i-motif stabilization. Molecular crowding, largely an excluded volume effect of added PEG, having a molecular weight in excess of 1,000 g/mol, appears to be responsible for stabilizing the more compact i-motif over the random coil at higher pH values.

H1.1 and H1.4 bind tightly to both short DNA oligomers and to CT-DNA (Ka≈1×10(7)). Binding is accompanied by an unfavorable enthalpy change (∆H≈+22 kcal/mol) and a favorable entropy change (-T∆S≈-30 kcal/mol). The Tm for the H1.4/CT-DNA complex is increased by 9 °C over the Tm for the free DNA. H1.4 titrations of the DNA oligomers yield stoichiometries (H1/DNA) of 0.64, 0.96, 1.29, and 2.04 for 24, 36, 48, and 72-bp DNA oligomers. The stoichiometries are consistent with a binding site size of 37±1 bp. CT-DNA titration data are consistent with binding site sizes of 32 bp for H1.1 and 36 bp for H1.4. The heat capacity changes, ΔCp, for formation of the H1.1 and H1.4/CT-DNA complexes are -160 cal mol(-1) K(-1) and -192 cal mol(-1)K(-1) respectively. The large negative ΔCp values indicate the loss of water from the protein DNA interface in the complex.

Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π-π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: K(TMPyP4)≈K(P)(5,15)>KP(5,10,15)>>>KP(5,10), KP(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an 'intercalation mode' while P(5,10,15) appears to interact by an 'end-stacking mode'.

Histone H1 is a chromatin protein found in most eukaryotes. ITC and CD have been used to study the binding of H1(0) and its C-terminal, H1(0)-C, and globular, H1(0)-G, domains to a highly polymerized DNA. ITC results indicate that H1(0) and H1(0)-C bind tightly to DNA (Ka≈1×10(7)), with an unfavorable ΔH (ΔH≈+22kcal/mol) and a favorable ΔS (-TΔS≈-30kcal/mol). Binding H1(0)-G to DNA at 25°C is calorimetrically silent. A multiple independent site model fits the ITC data, with the anomaly in the data near saturation attributed to rearrangement of bound H1, maximizing the number of binding sites. CD experiments indicate that H1(0)/DNA and H1(0)-C/DNA complexes form with little change in protein structure but with some DNA restructuring. Salt dependent ITC experiments indicate that the electrostatic contribution to binding H1(0) or H1(0)-C is small ranging from 6% to 17% of the total ΔG.

Elastin-like Polypeptides (ELPs) are genetically engineered biopolymers that are derived from the endogenous protein Tropoelastin. ELPs are structurally disordered and soluble at low temperatures but transition to a β-spiral and aggregate at a Transition Temperature (TT). This aggregation is being explored as a novel drug delivery vector by thermally targeting systemically delivered ELP-drug conjugates. We are investigating the biophysical properties of ELP[V5G3A2-150] as a means of understanding and predicting the behavior in vivo. We have investigated the hydrodynamic, structural and thermodynamic properties of ELP[V5G3A2-150] through the use of CD, turbidity, DLS, DSC and SV. DLS and SV analyses suggest that ELP[V5G3A2-150] experiences small amounts of weak association below the TT that increase with temperature. CD analyses further indicate that below the TT ELP[V5G3A2-150] consists of both disordered (≈75%) and β-conformation (≈25%) and as the temperature and concentration is increased the % β-conformation increases. The temperature & concentration dependence of β-conformation suggests that the weak association can be attributed to heterogeneous β-sheets. SV revealed that above the TT ELP[V5G3A2-150] exhibits a temperature dependent critical concentration (CC). This CC is consistent with the TT and suggests that the TT may be described as a solubility constant.Assembly in serum raises the TT by ≈ 2.5°C. This is opposite to the expected effect of macromolecular crowding and suggests that certain serum proteins may be associating with ELP[V5G3A2-150]. Investigation of this effect through SV was greatly complicated by the presence of the Johnston-Ogston (J-O) effect. Further investigation suggested additional complexity in systems exhibiting the J-O effect than previously reported. Two of the additional complexities already determined are cross-term hydrodynamic non-ideality and high-concentration convection. Additional research into the effects of attaching CPPs to ELP[V5G3A2-150] will be presented. Work supported by NSF ARRA 0959211 grant.