Development and validation of a high-performance liquid chromatography-fluorescence detection method for the accurate quantification of colistin in human plasma

Recently, colistin has become one of the most important drugs for treating infections caused by multidrug-resistant Gram-negative bacteria. Therapeutic drug monitoring is recommended to ensure the safety and efficacy of colistin and to improve clinical outcomes. This study developed an accurate and sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for the quantification of colistin in human plasma. The sample preparation included protein precipitation using trichloroacetic acid (TCA) and methanol, followed by in-solid phase extraction (In-SPE) derivatization with 9-fluorenylmethyl chloroformate (FMOC-C1). A Poroshell 120 EC-C18 2.1 x 100 mm (2.7 mu m) column was used in the HPLC method with a mobile phase composed of acetonitrile (ACN), tetrahydrofuran (THF), and deionized (DI) water (82%, 2%, 16% (v/v), respectively). Polymyxin B1 was used as the internal standard. The total analysis time was 22 min under optimal separation conditions. The HPLC-FLD method was validated over a therapeutic range of 0.3-6.0 mu g mL(-1). The intra-day and inter-day precisions for colistin A and colistin B were below 9.9% and 4.5% relative standard deviations, respectively. The accuracy test results were between 100.2 and 118.4%. The extraction recoveries were between 81.6 and 94.1%. The method was linear over the test range, with a 0.9991 coefficient of determination. The limit of detection was 0.1 mu g mL(-1). The validated HPLC-FLD method was successfully applied to quantify the colistin concentrations in 2 patient samples for therapeutic drug monitoring. (C) 2014 Elsevier B.V. All rights reserved

Abstract 423: Induced proximity and proteolytic targeting of LCK as a novel therapeutic approach in T-cell leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer, with a particularly dismal prognosis in patients with relapsed diseases. Our group recently discovered LCK dependency as a therapeutic vulnerability in 33% of T-ALL and LCK inhibitor dasatinib exhibited strong efficacy in vitro and in vivo (Nat Cancer 2, 284, 2021). However, the transient LCK inhibition by dasatinib resulted in only partial responses, and novel agents are needed to suppress LCK signaling in T-ALL in a sustained fashion to achieve long-term efficacy. We synthesized a set of proteolytic targeting chimeras (PROTACs) that bring LCK to cereblon (CRBN) E3 ligase for ubiquitination and degradation. LCK-PROTACs showed extremely high anti-leukemia sensitivity, i.e., up to 1,561.3 fold increase over dasatinib in LCK-activated T-ALL cell line. Complete LCK degradation was induced by most LCK-PROTACs. Based on solubility, permeability, stability, and anti-leukemia effects in vitro, we prioritized SJ001011646 for further evaluations. SJ001011646 induced 50% LCK degradation at the concentration at ~0.001nM, with superior cytotoxic effects in patient-derived primary T-ALL samples compared to dasatinib. In addition, we confirmed the formation of ternary complex of LCK, SJ001011646, and CRBN, using the AlphaLISA assay. In a wash-out experiment, LCK-dependent T-ALL cells were exposed to SJ001011646 or dasatinib for 18 hours and then monitored for viability recovery. Both dasatinib and SJ001011646 led to dramatic growth inhibition within 2 days. While dasatinib-treated cells started to recover 96 hours after drug removal, cells treated with SJ001011646 failed to recover even after 240 hours. To systematically identify therapeutic targets of SJ001011646, we performed proteomic profiling of T-ALL cells before and after drug treatment in vitro. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which LCK was most significantly reduced by PROTAC. In parallel, kinome-binding profiling confirmed LCK as a primary target of SJ001011646. Finally, we explored pharmacokinetic and pharmacodynamic properties of SJ001011646 in vivo, using patient-derived xenograft models of T-ALL. Given at the same dosage level as a single injection, SJ001011646 showed 367% increase in the duration of LCK suppression compared to dasatinib. Taken together, we described the development and preclinical evaluation of LCK-targeting PROTACs in T-ALL. Inducing proximity to CRBN and therefore degradation, these agents can produce sustained LCK suppression with much improved anti-leukemia efficacy than small molecule inhibitors, and are therefore promising novel therapeutic agents in T-ALL. Citation Format: Jianzhong Hu, Jamie Jarusiewicz, Jaeki Min, Lei Yang, Divyabharathi Chepyala, Marisa Actis, Guoqing Du, Brandon Smart, Dylan Maxwell, Boer Xie, Satoshi Yoshimura, Junming Peng, David T. Teachey, Zoran Rankovic, Jun J. Yang. Induced proximity and proteolytic targeting of LCK as a novel therapeutic approach in T-cell leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 423.</jats:p

Development of Proteolytic Targeting Chimeras to Target Lck in T-Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy for which novel therapies are much needed especially in patients with relapsed diseases. By combining large-scale ex vivo pharmacotype profiling with network-based systems biology analyses, our group recently identified LCK dependency as a therapeutic vulnerability in 44% of T-ALL in children (Nat Cancer 2, 284-299, 2021). LCK inhibitors such as dasatinib exhibit striking anti-leukemia effects in this T-ALL subset. However, the transient LCK inhibition by dasatinib only resulted in incomplete response to monotherapy unless the drug was delivered continuously at a high level. Therefore, it is imperative to develop novel agents that produce sustained suppression of LCK signaling in T-ALL. To this end, we synthesized a set of proteolytic targeting chimeras (PROTACs) that target LCK and cereblon (CRBN) E3 ligase. These PROTACs bind and recruit LCK to CRBN E3 ligase, rendering LCK susceptible to ubiquitination and ultimately proteasomal degradation. Cell viability assay was performed in an LCK-dependent T-ALL cell line KOPT-K1 to determine its sensitivity to this panel of PROTACs. PROTACs showed up to 6.9-fold improvement in cytotoxicity relative to dasatinib. To validate PROTAC-induced LCK degradation, KOPT-K1 cells were treated with PROTACs or dasatinib at 100 nM for 24 hours and LCK protein was quantified by western blotting. LCK degradation occurred rapidly in the presence of PROTAC agents whereas dasatinib treatment did not affect LCK abundance. Furthermore, PROTAC-induced apoptosis of T-ALL cells was abolished by lenalidomide, a CRBN binder, suggesting that their cytotoxic effects were mediated by CRBN-driven LCK degradation. We also validated these findings in patient-derived T-ALL samples. In addition, we determined solubility, permeability, and stability of these PROTACs in vitro. Based on anti-leukemia effects as well as physical chemical properties, we prioritized PROTACs SJ001011646 and SJ001011447 as our top candidates for further evaluations. We hypothesized that the catalytic protein degradation by PROTACs will produce a more sustained suppression of the LCK signaling compared to transient LCK inhibition by dasatinib. To test this, we performed a wash-out assay comparing anti-leukemic effects of top PROTACs with dasatinib. KOPT-K1 cells were treated with vehicle, dasatinib, SJ001011646 and SJ001011447, respectively at 100 nM for 18 hours. Cells were then washed and placed in drug-free culture with viability monitored daily. The vehicle treated cells exhibited an exponential growth while drug treated groups showed dramatic growth inhibition within 2 days. Notably, dasatinib-treated cells continued to undergo apoptosis for 96 hours after drug removal before started to recover. By contrast, SJ001011447 treatment repressed cell growth for 144 hours post wash-out. Most impressively, there was no evidence of growth recovery in cells treated SJ001011646 even 240 hours after drug removal. In addition, we confirmed the formation of ternary complex of LCK, PROTAC, and E3 ligase, using the AlphaLISA assay. To systematically identify therapeutic targets of PROTACs, we performed proteomic profiling of KOPT-K1 cells before and after drug treatment in vitro. Cells were treated with vehicle, SJ001011447 or SJ001011646 at 100 nM for 24 hours before harvested for Tandem Mass Tag-based proteomic profiling. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which 34 and 35 were significantly changed by SJ001011447 and SJ001011646, respectively (p&amp;lt;0.05, foldchange &amp;gt;2 or &amp;lt;-2). LCK was among proteins most significantly reduced after PROTAC treatment. Finally, we also developed formulation for PROTAC SJ001011646 for in vivo testing; and preclinical pharmacokinetic and pharmacodynamic characterization of this molecule is ongoing using T-ALL xenograft models. In conclusion, we developed LCK-targeting PROTACs with potent anti-leukemia effects. Highly effective in degrading LCK protein, these agents produced sustained LCK suppression superior to small molecule inhibitors, pointing to novel strategies to therapeutically target LCK in T-ALL. Disclosures No relevant conflicts of interest to declare. </jats:sec

Degradation of Janus kinases in <i>CRLF2</i>-rearranged acute lymphoblastic leukemia

Abstract CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT–driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.</jats:p

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators

International audienceWhereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins