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Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing
No full textMALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization
RIPK3-MLKL – Mediated neutrophil death requires concurrent activation of fibroblast activation protein alpha (FAP-α)
No full textCytokine-primed neutrophils can undergo a non-apoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF-primed human neutrophils. N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), a general serine protease inhibitor, completely blocked both cell death and CD44-mediated production of reactive oxidative species (ROS), suggesting that at least one serine protease is active in the death pathway proximal to NADPH oxidase activation. In subsequent experiments, we observed that several inhibitors known to block the enzymatic function of FAP-α were also able to block CD44-mediated ROS production and cell death, but not FAS receptor – mediated apoptosis. To understand how FAP-α is involved in this non-apoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3’-kinase (PI3K) and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL and p38 MAPK activation, but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis (RA) underwent a GM-CSF - independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3 – MLKL pathway activates NADPH oxidase, but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated non-apoptotic neutrophil death leading to tissue damage
Synthesis Development of the selective estrogen receptor degrader (SERD) LSZ102
No full textThe development of the synthesis of the selective estrogen receptor degrader (SERD) drug candidate LSZ102 from the research synthesis to late phase development synthesis is described in this article. The synthesis of LSZ102 could be significantly improved in regards of overall yield, removal of all chromatographic purifications and number of steps by modifying the original disconnection strategy from a Pd catalyzed Suzuki coupling to a Pd catalyzed conversion of an aryl bromide to a phenol and a Pd catalyzed C-H activation as key step.
The same content will be also shown at 3rd Edition of Unite Scientific Chemistry Conference (USCC-2023) to be held during June 22-23, 2023 in Rome, Italy and two online seminars in March 2021. The corresponding slides are in the upload section
Cryoconcentration and 3D Temperature Profiles during Freezing of mAb Solutions in Large-Scale PET Bottles and a Novel Scale-Down Device
No full textPurpose Small-scale models that simulate large-scale freezing of bulk drug substance of biopharmaceuticals are highly needed to define freezing and formulation parameters based on process understanding. We evaluated a novel scale-down device (SDD), which is based on a specially designed insulation cover, with respect to changes in concentration after freezing, referred to as cryoconcentration, and 3D temperature profiles. Furthermore, the effect of the initial monoclonal antibody (mAb) concentration on cryoconcentration was addressed.
Methods 2 L and 125 mL bottles were utilized. Temperatures were mapped using type T thermocouples. Frozen blocks were cut before mAb and histidine concentrations were analysed by HPLC. In addition, concentration- and temperature-dependent viscosities were measured.
Results 3D freezing profiles in the SDD were comparable to large-scale bottles. The SDD accurately predicted cryoconcentration of both mAb and histidine of large-scale freezing. Concentric changes in concentration were evident as well as an unforeseen diluted core at the last point to freeze. At low initial mAb concentration cryoconcentration was substantial, while high initial mAb concentration suppressed cryoconcentration almost completely.
Conclusion The novel SDD gives detailed insights into large-scale freezing of mAb solutions using only a fraction of the simulated volume. It is a promising material- and cost-saving tool to understand large-scale freezing processes
Two RNA tunnel inhibitors bind in highly conserved sites in Dengue virus NS5 polymerase: structural and functional studies.
No full textDengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. Whilst a number of allosteric inhibitors have been reported for Hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flow-chart combining biochemical and biophysical approaches, two DENV RdRp non-nucleoside inhibitors were identified and characterized. These inhibitors show low to high micro-molar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the finger and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the finger loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in Flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition
Discovery of Novel Antibiotics as Covalent Inhibitors of Fatty Acid Synthesis
No full textThe steady increase in the prevalence of multidrug-resistant Staphylococcus aureus has urged the search for novel antibiotics to combat this clinically important pathogen. In an effort to discover antibacterials with new chemical structures and mechanisms, we performed a growth inhibition screen of a synthetic library against Staphylococcus aureus and discovered a promising scaffold with 1,3,5-oxadiazin-2-one core. These compounds are potent against both methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains. Isolation of compound resistant strains followed by whole genome sequencing revealed its cellular target as FabH, a key enzyme in bacterial fatty acid synthesis. Detailed mechanism of action studies suggested the compounds inhibit FabH activity by covalently modifying its active site cysteine residue with high selectivity. A crystal structure of FabH protein modified by a selected compound Oxa1 further confirmed covalency and suggested a possible mechanism for reaction. Moreover, the structural snapshot provided an explanation for compound selectivity. Based on the structure, we designed and synthesized Oxa1 derivatives and evaluated their antibacterial activity. The structure-activity rela-tionship supports the hypothesis that non-covalent recognition between compounds and FabH is critical for the activity of these covalent inhibitors. We believe further optimization of the current scaffold could lead to antibacterial with potentials to treat drug resistant bacteria in the clinic
Characterization of membrane adsorbers used for impurity removal during the continuous purification of monoclonal antibodies
No full textDownstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed
Toward a scalable synthesis and process for EMA401. Part III: Using an engineered phenylalanine ammonia lyase enzyme to synthesize a non-natural phenylalanine derivative
No full textA process using engineered phenylalanine ammonia lyase (PAL) enzymes was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of the manuscript, the detailed results from a screening for the optimal reaction conditions are presented, followed by the discussion of several work-up strategies investigated. In the PAL catalyzed reaction, 70–80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet-Spengler reaction with formaldehyde and the corresponding tetrahydroisoquinoline derivative was isolated in 60–70% yield with >99.9:0.1 er. Based on our screenings, carbonate/carbamate buffered ammonia at 9–10 M NH3 concentration and pH 9.5–10.5 were found as the optimal conditions. Enzyme loadings down to 2.5wt% (E:S 1:40 w/w) could be achieved and substrate concentrations between 3–9 v/w (1.17–0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature-dependence of the entropy term with subsequent cooling to 20 °C and achieving maximum conversion. This temperature gradient also allowed balancing enzyme stability (low at 45 °C, high at 20 °C) with activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various work-up operations investigated, a sequence consisting of denaturation of the enzyme, followed by NH3/CO2 removal by distillation, acidification and telescoping to the subsequent Pictet-Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter and more cost effective alternative to the previous process
Is there a role for the No Observed Adverse Effect Level in safety pharmacology?
No full textIn nonclinical toxicology the highest dose or exposure without test article-related adverse effects, known as the No Observed Adverse Effect Level (NOAEL), is a variable that may be determined. In safety pharmacology the vast majority of the endpoints measured are quantitative numeric functional endpoints such as changes in heart rate, blood pressure or respiratory frequency, endpoints that are usually not assessed using a defined framework of adversity. Therefore, we asked the question: is there a role for the NOAEL in safety pharmacology? To populate our consideration, we conducted a survey via the Safety Pharmacology Society. We found that within safety pharmacology there is no formal definition of adversity and no guidance on defining NOAEL. We also found, perhaps unsurprisingly, there is no agreed rubric for using a NOAEL in safety pharmacology and we learned that the NOAEL is not a requirement in order to progress a new investigational drug through the regulatory process. Thus, a summary label such as NOAEL lacks nuance and disregards context in relation to the nature and the severity of the safety pharmacology findings. Consequently, defining ‘adversity’ and determining a NOAEL in safety pharmacology studies are not recommended since the range of functional endpoints investigated do not conform to a binary ‘toxic/non-toxic’ rubric. Focusing on describing test article-related effects on safety pharmacology endpoints, using reasoned arguments as part of an integrated risk assessment, will ensure that the clinical pharmacologists and regulatory bodies see a clear description of relevant findings at each dose or exposure level. This will inform a narrative about the potential risks of the test article and support a recommendation for optimal dose setting and optimal monitoring of vital signs in clinical trial
Estimating Shelf Life Through Tolerance Intervals
No full textThis paper is a continuation of the research published by the Stability Shelf Life
Working Group as chartered under the Product Quality Research Institute. The
Working Group was formed in 2006 and disbanded in late 2019. Following the
philosophy presented by the Working Group on how to characterize the stability shelf
life paradigm (Capen et al., 2012), shelf life is estimated here in terms of defining risk
as a specified proportion of the pharmaceutical stability distribution of interest being out
of specification. Shelf life can be defined for the batch mean distribution for regulatory
issues, as well as for the product distributions for patient interests. Estimates of shelf
life are proposed corresponding to each stability distribution through the use of
statistical tolerance intervals. Appropriate estimates of the between‑batch and
within‑batch variance components are obtained through a random coefficient mixed
regression model analysis based on the best fit to batch stability response data.
Tolerance interval estimates are computed as part of the mixed model analysis and
computed directly using the statistical definition of the stability distributions. A proposed
rationale is offered on how to select an appropriate proportion allowed out of
specification to define a meaningful shelf life. Examples of the proposed shelf life
estimates are presented using industry stability batch data. For each example, the
traditional ICH shelf life estimate is given for comparison