E7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. To ...understand their mechanism of action, we performed kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of the RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other proteins of interest.
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•DCAF15-sulfonamide binds to RBM39 through a single α helix of RBM39•The structure reveals how a sulfonamide molecular glue recruits RBM39 to DCAF15•Both RBM39 and E7820 have weak affinity to DCAF15 individually•Synergistic binding to DCAF15 leads to the formation of stable ternary complex
Du et al. describe in structural and kinetic detail how a sulfonamide glue, E7820, recruits RBM39 to DCAF15, a CUL4-related E3 ubiquitin ligase. RBM39 and E7820 each has weak affinity to DCAF15; however, synergistic binding of the two to DCAF15 leads to the formation of a stable ternary complex.
The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clinical ...activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metabolism of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by reduction of phase 2 metabolism. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clinical pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clinical activity shows promise for PT2977 in the treatment of ccRCC.
Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant ...effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.
Sex-specific equations for predicting maturity offset, time before or after peak height velocity (PHV), were evaluated in 63 girls and 74 boys from the Fels Longitudinal Study. Serially measured ...heights (0.1 cm), sitting heights (0.1 cm), weights (0.1 kg), and estimated leg lengths (0.1 cm) from 8 to 18 years were used. Predicted age at PHV (years) was calculated as the difference between chronological age (CA) and maturity offset. Actual age at PHV for each child was derived with a triple logistic model (Bock-Thissen-du Toit). Mean predicted maturity offset was negative and lowest at 8 years and increased linearly with increasing CA. Predicted ages at PHV increased linearly with CA from 8 to 18 years in girls and from 8 to 13 years in boys; predictions varied within relatively narrow limits from 12 to 15 years and then increased to 18 years in boys. Differences between predicted and actual ages at PHV among youth of contrasting maturity status were significant across the age range in both sexes. Dependence of predicted age at PHV upon CA at prediction and on actual age at PHV limits its utility as an indicator of maturity timing and in sport talent programs.
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Recent years have seen a resurgence in drug discovery efforts aimed at the identification of covalent inhibitors which has led to an explosion of literature reports in this area and ...most importantly new approved therapies. These reports and breakthroughs highlight the significant investments made across the industry in SAR campaigns to optimize inhibitors. The potency of covalent inhibitors is generally considered to be more accurately described by the time-independent kinetic parameter kinact/Ki rather than a by a simple IC50 since the latter is a time-dependent parameter. Enzyme substrate concentrations are an additional important factor to consider when attempting to translate parameters derived from enzymology experiments to phenotypic behavior in a physiologically relevant cell-based system. Theoretical and experimental investigations into the relationship between IC50, time, substrate concentration and Kinact/Ki provided us with an effective approach to provide meaningful data for SAR optimization. The data we generated for our JAK3 irreversible covalent inhibitor program using IC50 values provided by enzyme assays with long incubations (>1h) coupled with physiological substrate concentration provided the medicinal chemist with optimal information in a rapid and efficient manner. We further document the wide applicability of this method by applying it to other enzymes systems where we have run covalent inhibitor programs.
HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the ...overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel-Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π*
interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC
= 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.
HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the ...overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel–Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π*Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.
The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary ...effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with lipid nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called charge-altering releasable transporters (CARTs). Using these inherently nonimmunogenic vehicles, we can tailor the vaccine immunogenicity by inclusion of coformulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of receptor binding domain (RBD)-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long-lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.
Charge-altering releasable transporters (CARTs) are a class of oligonucleotide delivery vehicles shown to be effective for delivery of messenger RNA (mRNA) both in vitro and in vivo. Here, we ...exploited the chemical versatility of the CART synthesis to generate CARTs containing the small-molecule drug fingolimod (FTY720) as a strategy to increase mRNA delivery and expression in lymphocytes through a specific ligand–receptor interaction. Fingolimod is an FDA-approved small-molecule drug that, upon in vivo phosphorylation, binds to the sphingosine-1-phosphate receptor 1 (S1P1), which is highly expressed on lymphocytes. Compared to its non-fingolimod-conjugated analogue, the fingolimod-conjugated CART achieved superior transfection of activated human and murine T and B lymphocytes in vitro. The higher transfection of the fingolimod-conjugated CARTs was lost when cells were exposed to a free fingolimod before transfection. In vivo, the fingolimod-conjugated CART showed increased mRNA delivery to marginal zone B cells and NK cells in the spleen, relative to CARTs lacking fingolimod. Moreover, fingolimod-CART-mediated mRNA delivery induces peripheral blood T-cell depletion similar to free fingolimod. Thus, we show that functionalization of CARTs with a pharmacologically validated small molecule can increase transfection of a cellular population of interest while conferring some of the targeting properties of the conjugated small molecule to the CARTs.
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