The central dogma of biology states that genetic information describes the flow ofinformation from DNA to RNA and then finally resting in proteins. The fundamental aspect that underlies all aspects of life is the expression and modification of proteins. One can even argue that there is no life without proteins. As such, many human diseases are either directly or indirectly related to dysfunction of proteins and can potentially be solved through protein therapeutics. Consequently, scientists have begun to harness the diversity of proteins to treat the diseases which plague man in the form of protein therapeutics such as clotting factors, cytokines, and growth factors. Unfortunately, the short circulation half-life of proteins is a major limiting factor which must be overcome before their widespread adoption as a platform for therapeutic development. Contributing factors to this short circulation half-life include renal elimination, proteasomal degradation, and metabolism in the liver. Namely, renal elimination is the main challenge for protein therapeutics and warrants clinicians to resort to higher doses and more frequent administrations to maintain necessary concentrations in the body. Unfortunately, side effects of this approach are dose limiting toxicities and reduced therapeutic outcomes as drug concentrations fluctuate drastically. As a result, addressing the challenge of renal elimination for protein therapeutics would allow for the development of novel treatments which were previously not viable. The human glomerulus readily filters out any particle smaller than approximately 30 kDa in weight. As a result, strategies adopted all share a common theme of endowing the protein with a greater effective size without compromising their natural activity against the intended target. The current approaches include conjugation to a polymer (i.e., PEGylation), covalent or non-covalent binding to a larger protein, or conjugating to the neonatal Fc receptor. Major limitations of these approaches include compromised activity caused by steric hinderance rooted inconjugation to moieties of larger size. This issue applies to all of the aforementioned reported approaches wherein activity becomes reduced, thus necessitating higher dosages. Furthermore, other limitations also exist such as humoral immune responses against polymers through anti-PEG antibodies, occurrence of organ damage, and solubility issues. A novel approach was recently developed by the Alhamadsheh lab which demonstratedthe ability of a small molecule linker termed “Transthyretin Ligand for Half-life Extension” (TLHE) to extend the circulation half-life of Gonadotropin releasing hormone. Most essentially, this was accomplished without compromising the potency or introducing a major sterically bulky group to the original peptide. Furthermore, additional concerns such as solubility issues was demonstrated to not be an issue either. In this work, human Interleukin-2 (IL-2) was chosen as a proof of concept to demonstrate application of the TLHE technology in a protein to address the aforementioned half-life challenge. Previously, a mixture of IL-2 and TLHE-IL-2 was demonstrated to maintain comparable activity to control IL-2 in both in vitro and ex vivo efficacy assays. Furthermore, a pharmacokinetic evaluation in rodents demonstrated significant half-life extension of TLHE-IL-2. The objective of this work was to shift the ratio of the IL-2/TLHE-IL-2 mixture to majority TLHE-IL-2 and or enhance the yield of the mixture for further in vivo efficacy evaluation. What would happen to a viral infection if suddenly there were a billion fake receptors forevery real target receptor. A version of this question is what led to the development of a novel HIV therapy at Duke University around 1996. Fast forward more than 30 years, man still lacks the proper tools to combat viral infections. One can argue that the Achilles’ heel of viral infections is their need to bind a specific receptor. This protein-protein interaction between viral proteins and human receptors is arguably the fundamental point behind all viral infections. Recently, the COVID-19 pandemic again challenged man to develop new weapons at a revolutionary pace in order to save lives. During this time, the Pentelute lab at Massachusetts Institute of Technology reported a humanized version of the peptide sequence thought to represent the binding face of the human ACE2 receptor41. The 23 amino acid sequence was derived from the α1 helix of ACE2 peptidase domain and referred to as, spike-binding peptide 1 (SBP1). It was this sequence which was postulated to be responsible for binding the receptor binding domain of the notorious COVID-19 spike protein. However, a major limitation of peptide is their short in vivo half-life (through serum proteases and renal filtration). Therefore, the main aim of our proposed research was to employ the TLHE approach to extend the in vivo half-life of the SBP1 peptide. This would allow the creation a COVID-19 entry inhibitor that could help combat the COVID-19 pandemic.

Gamma-hydroxybutyric acid (GHB) is an endogenous shorty chain fatty acid that is used clinically as Xyrem to treat narcolepsy. GHB is best known for its illicit use and abuse due to its sedative/hypnotic and euphoric effects. It is used in body building, for recreational use and sexual assault. Nonlinear toxicokinetics of GHB has been described in humans and rats with decreased total clearance at higher doses due to capacity-limited metabolism resulting in a higher plasma exposure. Renal clearance increases with increasing dose and becomes the major route of GHB elimination in overdose cases due to saturation of GHB metabolism. Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) have been identified as major transporters in the renal reabsorption of GHB moving it from filtrate back to systemic circulation. Our laboratory has previously investigated sex differences in GHB toxicokinetics at 600 mg/kg in rats and identified sex differences in MCT expression in the liver and kidney. These data suggest that individual sex hormones may be involved in altering MCT and SMCT expression in drug disposition tissues and as a result alter GHB toxicokinetics. The present study had three objectives:1) GHB toxicokinetics were evaluated over the estrous cycle, and in the presence and absence of sex hormones following a dose of 1000 mg/kg iv in rats; 2) The role of individual female sex hormones on altering GHB toxicokinetics was investigated at 1000 mg/kg and 1500 mg/kg iv following sex and cross-sex hormone treatment with 17β-estradiol and progesterone, alone or in combination; and 3) renal MCT1, MCT4, SMCT1 and CD147 mRNA and membrane protein expression were quantified in response to female sex and cross-sex hormone treatment. We have demonstrated that GHB toxicokinetics and renal clearance vary between sexes, over the estrus cycle in females and in the absence of female and male sex hormones. In hormone-treated animals, GHB toxicokinetics were altered following sex and cross-sex hormone treatment with significantly increased total clearance and decreased GHB plasma exposure at 1000 mg/kg. Significant differences in renal and metabolic clearance were observed following 1000 mg/kg and 1500 mg/kg GHB suggesting altered regulation of the underlying clearance pathways. Additionally, we have investigated the renal mRNA and membrane-bound protein expression of MCT1, MCT4, CD147 and SMCT1 which were significantly altered in response to female sex hormones in both OVX (ovariectomized female rats, ovary removal surgery performed) and CST (castrated male rats, testicles removal surgery performed) rats. The mechanisms underlying MCT/SMT regulation by female sex hormones appear to vary based on the specific transporter. The alteration of renal monocarboxylate transporters in response to female sex hormones may contribute to the observed differences in GHB toxicokinetics, which may benefit the potential antidotes of GHB as a combined therapeutic strategy in clinic. In future, inhibition studies should be performed with the coadministration of MCTs/SMCTs inhibitor with GHB to further confirm the contribution of monocarboxylate transporters to GHB toxicokinetics following sex and cross-sex hormone treatment. The influence of female sex hormones on GHB-related metabolic enzymes and monocarboxylate transporters in liver should be evaluated to further explore the mechanism of underlying the observed alterations in metabolic clearance. Sex hormone receptor expression should be evaluated by western bolt and correlated with transporter expression, combined with analysis of sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further elucidate the mechanisms underlying MCTs/SMCTs regulation in response to female sex hormones. Additionally, GHB TK studies should also be conducted, combined with sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further confirm the effect of sex hormone receptor antagonist in GHB toxicokinetics. The MCT/SMCT expression is a key determinant of their substrates’ drug disposition; sex differences and altered regulation in response to sex and cross-sex hormone treatment may contribute to differences in GHB toxicokinetics and toxicity.

The aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, has been acknowledged as a critical regulator of xenobiotic-induced toxicity and carcinogenesis. In the absence of ligand, the AHR is cytoplasmic in a complex with Hsp90, p23, XAP2, and Src. The AHR complex translocates to the nucleus upon ligand binding. After releasing its chaperones, it forms a heterodimer with ARNT, which subsequently binds to a dioxin-responsive element (DRE) for target genes transcription. Multiple aspects of cells are altered by the substantial expression of AHR target genes. Even without the AHR ligand, the cytoplasmic AHR plays a critical role in tumor progression by affecting various cellular functions. Thus, understanding the mechanisms of AHR degradation is crucial, which provides novel ways to control the AHR target genes transcription and cellular functions. In addition to the 26S proteasomal degradation triggered by ligand or geldanamycin treatment, we discovered a novel AHR degradation pathway mediated by autophagy-lysosome in A549 cells. Specifically, the chaperone-mediated autophagy (CMA) facilitates the degradation of basal AHR in the lysosome. It can be activated by 6-AN, resulting in downregulated AHR protein levels and functions, including the ligand-dependent target genes transcription and cell migration/invasion process in A549 cells.p23 as a part of the AHR cytoplasmic complex has been continuously studied in our lab over the past decade. The most prominent role of p23 is protecting AHR from degradation in both immortalized cancer cell lines (mouse hepatoma Hepa1c1c7, human hepatoma Hep3B, human cervical HeLa) and untransformed human lung bronchial/tracheal epithelial (HBTE) cell lines. It encouraged us to investigate the mechanisms further. In A549 cells, downregulation of p23 content reduced AHR protein levels, partially due to an elevated AHR protein degradation. This degradation was not reversed by proteasome inhibitor MG132 but partially restored by lysosome inhibitor CQ. We cannot rule out the possibility that selective macroautophagy was involved in the basal AHR degradation in A549 cells since the PLA results showed a positive interaction between AHR and LC3B. So far, Hela cells could be the best expression system for HaloTag-AHR overexpression. Thus, we can use the HaloTag technology as a powerful tool to study the AHR degradation mechanism via protein labeling and LC-MS/MS analysis.

Drug delivery using liposomes is a promising technology that has the potential to deliver drugs to the site of action. Stability of liposomal drug delivery system under physiological conditions plays a crucial role in achieving desired therapeutic efficacy. Stability of the liposomal delivery systems can be determined by performing certain in-vitro studies which have the ability to predict in-vivo stability of liposomes. The aim of our study was to evaluate current available in-vitro methods that are used in predicting stability of liposomal drug delivery systems to determine which one of them is a better predictor of in-vivo stability of liposomes. Our second aim was to evaluate the in-vitro stability of the pHsensitive liposomes (contains morpholine based pH-sensitive lipid MOR) that were developed by our group. Liposomes with different lipid compositions were prepared using thin film hydration method to perform protein adsorption study. Since serum proteins adsorb on to liposomes surface (which might include substantial amount of opsonins that can trigger immune response against liposomes) upon intravenous administration, in-vitro protein adsorption Study is used as an indicator of in-vivo circulation half-life of liposomal carrier. In-vitro protein adsorption study includes two major Steps, first step is separation of liposomes with adsorbed protein from unadsorbed serum proteins and the second step is to evaluate the adsorbed protein quantitatively as well as qualitatively. The separation of liposomes from serum was achieved through size exclusion chromatography (SEC) using sepharose CL-4B gel. From the results obtained and upon discussion with vendors of sepharose gel we concluded that SEC using sepharose cl-4b is not a suitable method to separate liposomes from serum, because the stationary phase can interact with liposomes and lipoproteins. The second in-vitro method studied was drug release from liposomes. Doxorubicin loaded liposomes with different lipid compositions were prepared to perform drug release assay. An indirect method (where drug retained inside liposomes was measured) developed by our group was used, which takes the advantage of interaction between dowex resin and doxorubicin. Upon comparing the drug release study data obtained with previously reported in-vivo data we observed that drug release assay has the potential to predict invivo stability of liposomes. From the in-vitro drug release study data it was observed that introduction of the pH-sensitive lipid compromised the stability of pegylated liposomes.

<p> Background</p><p> Approximately half of adults in the U.S. fit the criteria for use of anti-obesity pharmacotherapy, but only 2% of those receive such treatment. This is in sharp contrast to the 8.4% of adults diagnosed with diabetes, with 86% of those receiving anti-diabetes pharmacotherapy. In 2012-2014, the first medications in 13 years were FDA approved for long-term management of obesity. In 2013-2014, the newest class of anti-diabetes pharmacotherapy, subtype 2 sodium-glucose transport protein inhibitors (SGLT2s), were FDA approved.</p><p> Methods</p><p> A retrospective analysis of extracted data from the IMS Health National Prescription Audit&trade; and Xponent&trade; assessed adoption rates of anti-obesity pharmacotherapies and SGLT2s using univariate linear regressions. Volumes of new and continuing prescriptions were compared using ratio analyses. Prescriber groups were compared by descriptive proportions according to prescription volumes, medical specialty, geographic region, and prescriber-drug overlap. </p><p> Results</p><p> The entire anti-diabetes market was 15 times the entire anti-obesity market. The anti-obesity market share was: 74.0% phentermine and 18.6% new anti-obesity pharmacotherapies. The mean increase in prescriptions per month were: 25,259 for SGLT2s (95% CI 23,133-27,383 p&lt;.0001), 5,154 for new anti-obesity pharmacotherapies (95% CI 4,800-5,507 p&lt;.0001), and 2,718 for phentermine (95% CI 1,345-4,089 p=0.0003). Medical specialties prescribing the majority of the analysis medications were Family Medicine/General Practice and Internal Medicine. Endocrinology had the highest prevalence of prescribers of any sub-specialty.</p><p> Conclusions</p><p> The adoption rate of SGLT2s was nearly exponential, while the adoption rate of new anti-obesity pharmacotherapies was linear. Considering the relative prevalence of obesity to diabetes and that obesity is a major cause of diabetes, these results are paradoxical and suggest biases against the prescribing of anti-obesity pharmacotherapies. The under-prescribing of anti-obesity pharmacotherapies is widely acknowledged, but this is the first prescription data to demonstrate its extent in the U.S.</p>

Protein kinases constitute essential biological and target class owing to the vital function of reversible phosphorylation catalyzed by these enzymes. With more than 500 kinases in the human genome, containing conserved structure and overlapping function, pose challenging targets for inhibition. Alternative methods for targeting protein kinases remain warranted as the traditional methods are biased toward ATP-competitive compounds. These methods have yielded successful therapeutics, however toxicity due to nonselectivity and limited development potential due to intense drug discovery efforts renders alternative modes of action attractive as new goals for protein kinase inhibition.Herein is presented a bivalent methodology for targeting protein kinases comprising staurosporine tethered a phage display cyclic peptide library such that the cyclic peptide is directed to areas on the kinase surface distinct from the ATP-site where staurosporine is bound. Presented in detail is this strategy as it was successfully applied to Protein Kinase A and the subsequent analysis of bivalent ligands. Since this initial study several kinases have been targeted with this methodology and Application to Aurora Kinase A will be explored in detail. An essential analysis of results to date is included as it applies to the redesign, construction, and application of new cyclic phage libraries. Finally, to complete the first successful application against Protein Kinase A, we explore kinase expression for structural studies.

<p> Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide in both men and women. Of relevance to our research presented herein are the Transforming growth factor &beta; (TGF-&beta;) signaling pathways and the heat shock response in the context of NSCLC. Dysregulation of TGF-&beta; signaling often results in disease and is a common feature of many cancers including NSCLC where it governs cell fate and epithelial plasticity through the epithelial-to-mesenchymal transition (EMT). Another key feature of oncogenic TGF-&beta; signaling is the crosstalk with other oncogenic pathways, like the NOTCH signaling pathway, which aids to restrict differentiation and modulates proliferation. Our research identified a mechanistic link between histone deacetylase 6 (HDAC6) and TGF-&beta;1-induced Notch1 signaling. When HDAC6 is knocked down with siRNA or its deacetylase function is pharmacologically inhibited TGF-&beta;1 activation of Notch signaling is abrogated. Within this paradigm we identified a protein complex consisting of HDAC6, heat shock protein 90 (HSP90), and the Notch1 receptor. In response to TGF-&beta;1 stimulation, HDAC6 rapidly deacetylates HSP90 at lysine 294 which corresponds with cleavage and activation of Notch1.</p><p> Our investigations also uncovered a unique feature of HSP90 function in NSCLC. Activation of the heat shock response triggers activation of Notch1 signaling. We demonstrated that HDAC6 regulates this heat shock-induced Notch1 signaling through modulation of HSP90 function of cytoplasmic sequestration of the key transcription factor that governs the heat shock response, heat shock factor 1 (HSF1). Brief exposure of NSCLC cells to 42&deg;C activates heat shock-induced Notch1 signaling, knockdown of HDAC6 with siRNA or pharmacological inhibition of HDAC6 abrogated this induction. In our investigations employing this combined strategy of targeting both HDAC6 and HSP90 we discovered that this treatment had an additive effect to enhance apoptotic markers and inhibit cell cycle progression in NSCLC cells. Individual HDAC6 or HSP90 inhibition slowed tumorigenesis and enhanced apoptosis of NSCLC in vivo. Taken altogether, our research identifies HDAC6 and HSP90 as regulators of key oncogenic pathways required for EMT and that combined inhibition of both these targets is a rational strategy to selectively kill NSCLC cells.</p>

Background: The current clinical practice of factor VIII (FVIII) prophylaxis revolves around converting patients with severe hemophilia A, hereafter simply referred to as hemophilia, phenotype (defined as plasma factor VIII coagulant activity [FVIII:C] <1 IU dL-1) to moderate hemophilia phenotype (defined as plasma FVIII:C from 1–5 IU dL-1). However, a wide inter-individual variation in bleeding tendency is observed despite changes in plasma FVIII:C (pharmacokinetic [PK] changes). Therefore, monitoring FVIII prophylaxis by global hemostasis biomarkers (pharmacodynamic [PD] response) can potentially be beneficial. Objective: To conduct appropriate PK/PD modeling using plasma FVIII:C and global hemostasis (platelet function and blood viscoelastic) biomarkers in severe hemophilia. Methods: Nine non-bleeding severe hemophiliacs (plasma FVIII:C <1 IU dL-1) with variant bleeding tendency (5 frequent bleeders and 4 infrequent bleeders) were infused with a recombinant factor FVIII (rFVIII) prophylactic dose (mean = 32.1 international units per kilogram [IU kg-1]). Blood was collected at baseline and 0.5-, 1-, 2-, 4-, 8-, 12-, 24- and 48-hours (h) post-dose for plasma FVIII:C, platelet function (platelet contractile force [PCF], clot elastic modulus [CEM] and force onset time [FOT]) and blood viscoelastic (reaction-time [R], kinetics-time [K] and maximum amplitude [MA]) biomarkers and activated partial thromboplastin time (aPTT). Non-compartmental analysis (NCA) was performed using standard methods. Compartmental analysis (CA) and PK/PD modeling were performed by non-linear regression. Correlation and analysis of variance (ANOVA) were used to explore the role of clinically relevant modifiers of bleeding tendency, as appropriate. ANOVA was used to assess inter-group differences in pertinent PK and PD parameters. P value <0.05 significance level was pre-specified for all statistical tests. Results: Mean (±SD) volume of distribution at steady state (Vss), total clearance (CLtot) and terminal half-life (t1/2) from NCA were 40.5 (±11.2) milliliter per kilogram (mL kg-1), 2.9 (±1.2) milliliter per hour per kilogram (mL h-1 kg-1) and 11.6 (±6.2) h, respectively. Mean (±SD) Vss and CLtot from the one-compartment body model (CA) were 39.6 (±8.9) mL kg-1 and 3.1 (±1.3) mL h-1 kg-1, respectively. The mean (±SD) baseline effect (E0) and slope from the PK/PD linear modeling were: for aPTT, 48.9 (±4.4) seconds (sec) and -0.025 (±0.009) second deciliter per international unit (sec dL IU-1), respectively; for PCF, 0.3 (±0.3) kilodynes (kdynes) and 0.008 (±0.004) kilodynes deciliter per international unit (kdynes dL IU-1), respectively; and for CEM, 0.0 (±0.0) kilodynes per square centimeter (kdynes cm-2) and 0.032 (±0.016) kilodynes deciliter per international unit per square centimeter (kdynes dL IU-1 cm-2), respectively. The mean (±SD) maximum effect (Emax) and half the maximum effective concentration (EC50) from the PK/PD sigmoidal Emax model were: for FOT, 70.1 (±16.9) % reduction and 87.8 (±31.4) IU dL-1 for FOT, respectively; for R, 74.9 (±26.0) % reduction and 68.5 (±28.4) IU dL-1, respectively; and for K, 73.2 (±36.4) % reduction and 67.2 (±29.0) IU dL-1, respectively. MA was not PK/PD modeled due to its low sensitivity. Conclusions: Plasma FVIII:C remained ≥1 IU dL-1 over the prophylactic interval. FOT and R were the most sensitive biomarkers at lower plasma FVIII:C. PCF and CEM were more sensitive than K and aPTT at lower plasma FVIII:C. MA was the least sensitive biomarker. Correlation and inter-group differences did not reach statistical significance (small sample size). These results may be used to assess risk of bleeding and dose-optimize FVIII prophylaxis in severe hemophilia.

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft into the presynaptic terminus and plays a critical role in maintaining the normal function of dopaminergic neurons. DAT is the major target of widely abused psychostimulant drugs, including cocaine and amphetamine. DAT also figures into disease states, and it is a target for therapeutic drugs. It is known that cathinone and methcathinone, β-keto analogs of amphetamine and methamphetamine, respectively, produce pharmacological actions similar to amphetamine. Cathinone and methcathinone analogs are recently gaining in popularity on the clandestine market (e.g. ‘bath salts’). Cathinone and methcathinone analogs as well as their amphetamine and methamphetamine counterparts were synthesized and examined at the hDAT expressed in Xenopus oocytes. One of the two major constituents of ‘bath salts’ (i.e., mephedrone) produced an electrophysiological signature similar to the dopamine releasing agent S(+)-amphetamine while the other major constituent (i.e., MDPV) produced an electrophysiological signature similar to the dopamine re-uptake inhibitor cocaine.

There continues to be a need for more effective analgesics. The α2-adrenoceptor (AR) agonist clonidine is an analgesic whose use is severely limited by undesirable side effects. meta-Chlorophenylguanidine (MD-354), an agent developed in our laboratory, selectively potentiates the antinociceptive effects of clonidine in a biphasic manner. Mechanistic studies suggest that both 5-HT3 receptor and α2-AR mechanisms are involved. To further evaluate mechanisms underlying the analgesia-potentiating effect of clonidine by MD-354, pharmacological studies using more established 5-HT3 receptor agonists: meta-chlorophenylbiguanide (mCPBG) and centrally-acting SR57227A, and non-selective α2-adrenoceptor ligand TDIQ, administered alone and in combination with clonidine, were conducted in mouse antinociceptive assays. None of the examined analogs produced an antinociceptive effect when administered alone. Nevertheless, mCPBG potentiated the antinociceptive actions of clonidine in a monophasic manner and the effect was antagonized by the 5-HT3 receptor antagonist tropisetron but not by tropisetron methiodide, suggesting that potentiation is, at least in part, due to a central 5-HT3 receptor mechanism. SR57227A did not alter the antinociceptive actions of clonidine. TDIQ was found to potentiate the analgesic actions of clonidine in a synergistic manner (as determined by an isobolographic analysis) and the effect was blocked by α2-AR antagonists (BRL-44408, imiloxan, ARC-239; α2A-, α2B-, and α2C-AR antagonists, respectively). This supports the hypothesis that MD-354 could be potentiating the analgesic actions of clonidine via an α2-AR agonist mechanism. In order to explore the role of the ring nitrogen atoms and the chloro substituent of conformationally-constrained rotamers of MD-354, analogs of 2-amino-7-chloro-3,4-dihydroquinazoline, with a varying number of nitrogen atoms in the ring were synthesized. Preliminary binding affinity results indicated that the ring nitrogen atoms are essential for 5-HT3 receptor binding. In attempt to explain the varied binding and functional activity of MD-354 at α2-ARs, 3D homology models of α2A-, α2B- and α2C-AR were generated and docking studies of the low-energy rotamers of MD-354 were conducted. The present studies support a role for the involvement of 5-HT3 receptors and α2-ARs in antinociception. Analgesic adjuvants with a dual mechanism of action such as MD-354 might represent a promising avenue to pain treatment.