Application of Thiol-yne/Thiol-ene Reactions for Peptide and Protein Macrocyclizations.

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated.

Inhibition of bcr-abl in human leukemic cells with a coiled-coil protein delivered by a leukemia-specific cell-penetrating Peptide.

The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML).1 Current therapies target the tyrosine kinase domain of Bcr-Abl, but resistance to these drugs is common.2 Bcr-Abl homo-oligomerization via its N-terminal coiled-coil (CC) domain is required for tyrosine kinase activity.3 Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a peptidomemetic known as CC(mut3), delivered as a plasmid.4 In this study, CC(mut3) is delivered to cells as a protein by utilizing a leukemia-specific cell-penetrating peptide (CPP).5 Here, recombinant CPP-CC(mut3) was expressed, purified, and tested for its antioncogenic activity. CPP-CC(mut3) was able to enter two leukemic cell lines (K562 and Ba/F3-P210) and inhibit Bcr-Abl activity as shown by induction of necrosis/apoptosis via 7-AAD/Annexin V staining, reduction of oncogenic potential in colony forming assays, reduction of cell proliferation, and inhibition of Bcr-Abl phosphorylation (kinase activity). Further, CPP-CC(mut3) did not enter nonleukemic cell lines (HEK293 and MCF-7). While CPP-CC(mut3) was able to enter the parental, nonleukemic Bcr-Abl(-) Ba/F3 pro-B cell line, it revealed no signs of activity in the assays performed, as expected. These results indicate the feasibility of using CPP-CC(mut3) as a therapeutic against CML.

Disruption of Bcr-Abl coiled coil oligomerization by design.

Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

A new oral testosterone (TLANDO) treatment regimen without dose titration requirement for male hypogonadism.

BACKGROUND: Male hypogonadism (testosterone level < 300 ng/dl) is a clinical syndrome that results from failure of the testis to produce physiological levels of testosterone. Most marketed testosterone replacement therapy products often require multiple dose adjustment clinic visits to achieve the desired, eugonadal testosterone levels. OBJECTIVE: To evaluate the efficacy and safety of a novel oral testosterone undecanoate therapy for the treatment of hypogonadism. MATERIAL AND METHODS: Ninety-five (N = 95) hypogonadal men were enrolled in this open-label, single-arm, multicenter study in the United States (NCT03242590). Subjects received 225 mg of oral testosterone undecanoate (TLANDO) twice a day for 24 days without dose adjustment. Primary efficacy was percentages of subjects who achieved mean 24-h testosterone levels within the eugonadal range and secondary efficacies were evaluated based on the upper limit of lab normal range of testosterone concentration. RESULTS: Subjects enrolled were on average age of 56 years, with about 17% of subjects older than 65 years. The mean body mass index was 32.8 kg/m2 . The baseline mean total testosterone values were below the normal range (202 ± 74 ng/dl). Post-treatment with 450 mg testosterone undecanoate daily dose without dose adjustment, 80% of subjects (95% confidence interval of 72%-88%) achieved a testosterone Cavg in the normal range and restored testosterone levels to mean testosterone Cavg of 476 ± 184 ng/dl at steady state. Testosterone restoration was comparable to other approved testosterone replacement therapy products. TLANDO was well tolerated with no deaths, no drug-related serious adverse events, and no hepatic adverse events. DISCUSSION AND CONCLUSIONS: TLANDO restored testosterone levels to the normal range in the majority of hypogonadal males. This new oral testosterone replacement therapy can provide an option for no-titration oral testosterone replacement therapy. This therapy has the potential to improve patient compliance in testosterone replacement therapy.

Investigating oral fluid and exhaled breath as alternative matrices for anti-doping testing: Analysis of 521 matched samples.

Current anti-doping testing is primarily conducted in urine and blood. Recently, due to confounding factors with urine and blood collections such as invasiveness, cost, and stringent shipping conditions, there has been a push for the use of alternative sample matrices to ameliorate these issues. Gaining support within the anti-doping field is the use of oral fluid, and more recently exhaled breath, as viable alternative or complementary matrices to traditional urine and blood for drug testing. Thus, we designed a first-in-field study with the purpose of investigating the utility of oral fluid and exhaled breath testing, and the preference of athlete participants, comparative to conventional anti-doping methods of urine testing. To accomplish this, 521 total matched samples, consisting of exhaled breath, oral fluid, and urine samples, were collected and analyzed, and the results compared across matrices. Participants in this study preferred the exhaled breath collection (rated 4.90 ±â€¯0.34 out of 5, mean ±â€¯SD) over the oral fluid collection procedure (4.29 ±â€¯0.85), and most preferred both over urine collections. Exhaled breath resulted in the shortest collection time (2.58 ±â€¯1.00 min, mean ±â€¯SD), followed by urine (3.08 ±â€¯1.50 min), and finally oral fluid (4.14 ±â€¯1.94 min). Prohibited substances from the drug categories of stimulants, narcotics, cannabinoids, diuretics, glucocorticoids, beta-blockers, and others, were analyzed in this study for a comparison of testing efficacy. Of the total findings 49% were detectable in only urine, 38% in urine + oral fluid, and 9% in all three matrices. Of the unique findings 3% were detectable in only oral fluid, 1% in oral fluid + breath, and 0% of unique findings were present only in exhaled breath. The findings from this study provide a strong foundation for the future use of oral fluid and exhaled breath as viable alternative or complementary matrices for in-competition anti-doping testing.

Computational Modeling of Stapled Peptides toward a Treatment Strategy for CML and Broader Implications in the Design of Lengthy Peptide Therapeutics.

The oncogenic gene product Bcr-Abl is the principal cause of chronic myeloid leukemia, and although several therapies exist to curb the aberrant kinase activity of Bcr-Abl through targeting of the Abl kinase domain, these therapies are rendered ineffective by frequent mutations in the corresponding gene. It has been demonstrated that a designed protein, known as CCmut3, is able to produce a dominant negative inactivating effect on Bcr-Abl kinase by preferentially oligomerizing with the N-terminal coiled-coil oligomerization domain of Bcr-Abl (Bcr-CC) to effectively reduce the oncogenic potential of Bcr-Abl. However, the sheer length of the CCmut3 peptide introduces a high degree of conformational variability and opportunity for targeting by intracellular proteolytic mechanisms. Here, we have examined the effects of introducing one or two molecular staples, or cross-links, spanning i, i + 7 backbone residues of the CCmut3 construct, which have been suggested to reinforce α-helical conformation, enhance cellular internalization, and increase resistance to proteolytic degradation, leading to enhanced pharmacokinetic properties. The importance of optimizing staple location along a highly tuned biological construct such as CCmut3 has been widely emphasized and, as such, we have employed in silico techniques to swiftly build, relax, and characterize a large number of candidates. This approach effectively allowed exploring each and every possible staple location along the peptide backbone so that every possible candidate is considered. Although many of the stapled candidate peptides displayed enhanced binding characteristics for Bcr-CC and improved conformational stability in the (Bcr-CC) bound form, simulations of the stapled peptides in the unbound form revealed widespread conformational variability among stapled candidates dependent on staple type and location, implicating the molecular replacement of helix-stabilizing residues with staple-containing residues in disrupting the native α-helical conformation of CCmut3, further highlighting a need for careful optimization of the CCmut3 construct. A candidate set has been assembled, which retains the native backbone α-helical integrity in both the bound and unbound forms while providing enhanced binding affinity for the Bcr-CC target, as research disseminated in this manuscript is intended to guide the development of a next-generation CCmut3 inhibitor peptide in an experimental setting.

Delivery of drugs and macromolecules to the mitochondria for cancer therapy.

Mitochondria are organelles that have pivotal functions in producing the energy necessary for life and executing the cell death pathway. Targeting drugs and macromolecules to the mitochondria may provide an effective means of inducing cell death for cancer therapy, and has been actively pursued in the last decade. This review will provide a brief overview of mitochondrial structure and function, how it relates to cancer, and importantly, will discuss different strategies of mitochondrial delivery including delivery using small molecules, peptides, genes encoding proteins and MTSs, and targeting polymers/nanoparticles with payloads to the mitochondria. The advantages and disadvantages for each strategy will be discussed. Specific examples using the latest strategies for mitochondrial targeting will be evaluated, as well as potential opportunities for specific mitochondrial compartment localization, which may lead to improvements in mitochondrial therapeutics. Future perspectives in mitochondrial targeting of drugs and macromolecules will be discussed. Currently this is an under-explored area that is prime for new discoveries in cancer therapeutics.

Resistant mutations in CML and Ph(+)ALL - role of ponatinib.

In 2012, ponatinib (Iclusig(®)), an orally available pan-BCR-ABL tyrosine kinase inhibitor (TKI) developed by ARIAD Pharmaceuticals, Inc., was approved by the US Food and Drug Administration for use in resistant or intolerant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). Ponatinib is the only approved TKI capable of inhibiting BCR-ABL with the gatekeeper T315I kinase domain mutation, known to be the cause for 20% of resistant or relapsed CML cases. In 2013, ponatinib sales were temporarily suspended due to serious side effects seen in nearly 12% of the patient population. These side effects are thought to stem from the potent nature and pan-activity of this TKI. ARIAD Pharmaceuticals, Inc. has since been permitted to resume sales and marketing of ponatinib to a limited patient population with an expanded black box warning. In the following review, the use of ponatinib in CML and Ph(+)ALL will be discussed. Mechanisms of resistance in CML are discussed, which provide insight and background into the need for this third generation TKI, followed by the molecular design and pharmacology of ponatinib, which lead to its success as a therapeutic. Finally, the efficacy, safety, and tolerability of ponatinib will be highlighted, including summaries of the important clinical trials involving ponatinib as well as its current place in therapy.

Basics and recent advances in peptide and protein drug delivery.

While the peptide and protein therapeutic market has developed significantly in the past decades, delivery has limited their use. Although oral delivery is preferred, most are currently delivered intravenously or subcutaneously due to degradation and limited absorption in the gastrointestinal tract. Therefore, absorption enhancers, enzyme inhibitors, carrier systems and stability enhancers are being studied to facilitate oral peptide delivery. Additionally, transdermal peptide delivery avoids the issues of the gastrointestinal tract, but also faces absorption limitations. Due to proteases, opsonization and agglutination, free peptides are not systemically stable without modifications. This review discusses oral and transdermal peptide drug delivery, focusing on barriers and solutions to absorption and stability issues. Methods to increase systemic stability and site-specific delivery are also discussed.