Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

A Randomized, Double-Blind, Placebo-Controlled Phase 3 Trial of Oral Brincidofovir for Cytomegalovirus Prophylaxis in Allogeneic Hematopoietic Cell Transplantation.

Cytomegalovirus (CMV) infection is a common complication of allogeneic hematopoietic cell transplantation (HCT). In this trial, we randomized adult CMV-seropositive HCT recipients without CMV viremia at screening 2:1 to receive brincidofovir or placebo until week 14 post-HCT. Randomization was stratified by center and risk of CMV infection. Patients were assessed weekly through week 15 and every third week thereafter through week 24 post-HCT. Patients who developed clinically significant CMV infection (CS-CMVi; CMV viremia requiring preemptive therapy or CMV disease) discontinued the study drug and began anti-CMV treatment. The primary endpoint was the proportion of patients with CS-CMVi through week 24 post-HCT; patients who discontinued the trial or with missing data were imputed as primary endpoint events. Between August 2013 and June 2015, 452 patients were randomized at a median of 15 days after HCT and received study drug. The proportion of patients who developed CS-CMVi or were imputed as having a primary endpoint event through week 24 was similar between brincidofovir-treated patients and placebo recipients (155 of 303 [51.2%] versus 78 of 149 [52.3%]; odds ratio, .95 [95% confidence interval, .64 to 1.41]; P = .805); fewer brincidofovir recipients developed CMV viremia through week 14 compared with placebo recipients (41.6%; P < .001). Serious adverse events were more frequent among brincidofovir recipients (57.1% versus 37.6%), driven by acute graft-versus-host disease (32.3% versus 6.0%) and diarrhea (6.9% versus 2.7%). Week 24 all-cause mortality was 15.5% among brincidofovir recipients and 10.1% among placebo recipients. Brincidofovir did not reduce CS-CMVi by week 24 post-HCT and was associated with gastrointestinal toxicity.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.

Efficacy of delayed brincidofovir treatment against a lethal rabbitpox virus challenge in New Zealand White rabbits.

In the event of a bioterror attack with variola virus (smallpox), exposure may only be identified following onset of fever. To determine if antiviral therapy with brincidofovir (BCV; CMX001) initiated at, or following, onset of fever could prevent severe illness and death, a lethal rabbitpox model was used. BCV is in advanced development as an antiviral for the treatment of smallpox under the US Food and Drug Administration's 'Animal Rule'. This pivotal study assessed the efficacy of immediate versus delayed treatment with BCV following onset of symptomatic disease in New Zealand White rabbits intradermally inoculated with a lethal rabbitpox virus (RPXV), strain Utrecht. Infected rabbits with confirmed fever were randomized to blinded treatment with placebo, BCV, or BCV delayed by 24, 48, or 72 h. The primary objective evaluated the survival benefit with BCV treatment. The assessment of reduction in the severity and progression of clinical events associated with RPXV were secondary objectives. Clinically and statistically significant reductions in mortality were observed when BCV was initiated up to 48 h following the onset of fever; survival rates were 100%, 93%, and 93% in the immediate treatment, 24-h, and 48-h delayed treatment groups, respectively, versus 48% in the placebo group (p < 0.05 for each vs. placebo). Significant improvements in clinical and virologic parameters were also observed. These findings provide a scientific rationale for therapeutic intervention with BCV in the event of a smallpox outbreak when vaccination is contraindicated or when diagnosis follows the appearance of clinical signs and symptoms.

The Role of Brincidofovir in Preparation for a Potential Smallpox Outbreak.

Smallpox (variola) virus is considered a Category A bioterrorism agent due to its ability to spread rapidly and the high morbidity and mortality rates associated with infection. Current recommendations recognize the importance of oral antivirals and call for having at least two smallpox antivirals with different mechanisms of action available in the event of a smallpox outbreak. Multiple antivirals are recommended due in large part to the propensity of viruses to become resistant to antiviral therapy, especially monotherapy. Advances in synthetic biology heighten concerns that a bioterror attack with variola would utilize engineered resistance to antivirals and potentially vaccines. Brincidofovir, an oral antiviral in late stage development, has proven effective against orthopoxviruses in vitro and in vivo, has a different mechanism of action from tecovirimat (the only oral smallpox antiviral currently in the US Strategic National Stockpile), and has a resistance profile that reduces concerns in the scenario of a bioterror attack using genetically engineered smallpox. Given the devastating potential of smallpox as a bioweapon, preparation of a multi-pronged defense that accounts for the most obvious bioengineering possibilities is strategically imperative.

The RAS/MAPK Axis Gets Stressed Out.

In this issue of Molecular Cell, Ritt et al. (2016) describe a stress-induced checkpoint that effectively suppresses RAS-MAPK signaling. This pathway, activated by agents such as Rigosertib that induce mitotic and oxidative stress, results in JNK-mediated inhibition of RAS-MAPK pathway components SOS and RAF.

Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the ß3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.

Tissue-Specific Mutations in BRAF and EGFR Necessitate Unique Therapeutic Approaches.

A predominant number of cancers are driven by mutations of key growth signaling genes. While it might be expected that the same alterations within a given oncogene would be identified in all tissues, there are clear cases of tissue specificity. Here, we highlight the tissue specificity of BRAF and EGFR alterations and implications for therapeutic targeting.

Effects of Obesity on Health Related Quality of Life Following Total Hip Arthroplasty.

Obesity is known to negatively impact health related quality of life (HRQoL). Although non-disease specific tools have been used to study HRQoL after THA in obese patients, these do not directly measure health utility improvements. All 435 THA patients in the current study, regardless of BMI, reported improvement in HRQoL as measured by EQ-5D, a universal, standardized, non-disease specific preference-based instrument. These data suggest obese patients value their quality of life improvement following THA as much as non-obese patients. Furthermore, the increased activity level observed following THA in obese patients suggests obese patients may also obtain non-disease specific benefits of a more active lifestyle. This information is important for future assessments of value and cost-effectiveness of THA in the obese population.

The efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: a model of smallpox disease.

Brincidofovir (BCV) has broad-spectrum in vitro activity against dsDNA viruses, including smallpox, and is being developed as a treatment for smallpox as well as infections caused by other dsDNA viruses. BCV has previously been shown to be active in multiple animal models of smallpox. Here we present the results of a randomized, blinded, placebo-controlled study of the efficacy and pharmacokinetics of a novel, "humanized" regimen of BCV for treatment of New Zealand White rabbits infected with a highly lethal inoculum of rabbitpox virus, a well characterized model of smallpox. Compared with placebo, a dose-dependent increase in survival was observed in all BCV-treatment groups. Concentrations of cidofovir diphosphate (CDV-PP), the active antiviral, in rabbit peripheral blood mononuclear cells (PBMCs) were determined for comparison to those produced in humans at the dose proposed for treatment of smallpox. CDV-PP exposure in PBMCs from rabbits given BCV scaled to human exposures at the dose proposed for treatment of smallpox, which is also currently under evaluation for other indications. The results of this study demonstrate the activity of BCV in the rabbitpox model of smallpox and the feasibility of scaling doses efficacious in the model to a proposed human dose and regimen for treatment of smallpox.

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis.

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C(Cdc20) substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding "ABBA motif" in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C(Cdc20) substrate destruction.

Naphthyridinone (NTD) integrase inhibitors 4. Investigating N1 acetamide substituent effects with C3 amide groups.

A series of N1 acetamide substituted naphthyridinone HIV-1 integrase inhibitors have been explored to understand structure-activity relationships (SAR) with various C3 amide groups. Investigations were evaluated using integrase enzyme inhibition, antiviral activity and protein binding effects to optimize the sub-structures. Lipophilicity was also incorporated to understand ligand lipophilic efficiency as a function of the structural modifications. Three representative analogs were further examined in a peripheral blood mononuclear cell (PBMC) antiviral assay as well as in vitro and in vivo drug metabolism and pharmacokinetic studies.

Carbamoyl pyridone HIV-1 integrase inhibitors 3. A diastereomeric approach to chiral nonracemic tricyclic ring systems and the discovery of dolutegravir (S/GSK1349572) and (S/GSK1265744).

We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (3) and S/GSK1265744 (4). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure-activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of 3 and 4, which are currently in clinical development.

Naphthyridinone (NTD) integrase inhibitors: N1 protio and methyl combination substituent effects with C3 amide groups.

Substituent effects of a series of N1 protio and methyl naphthyridinone HIV-1 integrase strand-transfer inhibitors has been explored. The effects of combinations of the N1 substituent and C3 amide groups was extensively studied to compare enzyme inhibition, antiviral activity and protein binding effects on potency. The impact of substitution on ligand efficiency was considered and several compounds were advanced into in vivo pharmacokinetic studies ultimately leading to the clinical candidate GSK364735.

The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation.

The fidelity of chromosome segregation depends on the spindle assembly checkpoint (SAC). In the presence of unattached kinetochores, anaphase is delayed when three SAC components (Mad2, Mad3/BubR1, and Bub3) inhibit Cdc20, the activating subunit of the anaphase-promoting complex (APC/C). We analyzed the role of Cdc20 autoubiquitination in the SAC of budding yeast. Reconstitution with purified components revealed that a Mad3-Bub3 complex synergizes with Mad2 to lock Cdc20 on the APC/C and stimulate Cdc20 autoubiquitination, while inhibiting ubiquitination of substrates. SAC-dependent Cdc20 autoubiquitination required the Mnd2/Apc15 subunit of the APC/C. General inhibition of Cdc20 ubiquitination in vivo resulted in high Cdc20 levels and a failure to establish a SAC arrest, suggesting that SAC establishment depends on low Cdc20 levels. Specific inhibition of SAC-dependent ubiquitination, by deletion of Mnd2, allowed establishment of a SAC arrest but delayed release from the arrest, suggesting that Cdc20 ubiquitination is also required for SAC inactivation.

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism.

BACKGROUND: Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the anaphase-promoting complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. RESULTS: Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20's C box. Cdc20 turnover by this intramolecular mechanism is cell cycle regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. CONCLUSION: We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle.

Combining symmetry elements results in potent naphthyridinone (NTD) HIV-1 integrase inhibitors.

A series of naphthyridinone HIV-1 integrase strand-transfer inhibitors have been designed based on a psdeudo-C2 symmetry element present in the two-metal chelation pharmacophore. A combination of two distinct inhibitor binding modes resulted in potent inhibition of the integrase strand-transfer reaction in the low nM range. Effects of aryl and N1 substitutions are disclosed including the impact on protein binding adjusted antiviral activity.