Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.

Abstract Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, but it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for RBD-ACE2 binding affinity assessments of ACE2 or RBD variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W along with multi-mutation variants as high-affinity candidates, which we also validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid clonal growth of myeloid lineage cells, with accumulation of immature blasts. Overall survival of AML patients is low and resistance to frontline chemotherapy is a major cause of treatment failure, highlighting the need for new therapies. It has been reported that AXL, a TAM family receptor tyrosine kinase, along with its ligand growth arrest-specific gene 6 (GAS6), mediates proliferation and survival of many types of cancer cells. An increasing body of evidence suggests that the GAS6/AXL pathway plays a major role in resistance to targeted therapies and conventional cytotoxic drugs. Particularly, increased expression levels of AXL and GAS6 have been reported in some AML and chronic myeloid leukemia (CML) patients, which are associated with poor prognosis. Several AXL inhibitors have recently been developed and some of them are in clinical trials. However, many of these small molecules are multi-kinase inhibitors and off-target effects and/or toxicity on healthy hematopoietic cells remain challenging. Moreover, it is still not known if the GAS6/AXL pathway is specifically activated in subgroups of AML patients carrying specific chromosomal abnormalities or mutations, and if AXL inhibitors can sensitize AML stem/progenitor cells to chemotherapy drugs and targeted therapeutics, since these cells are highly resistant to current anti-cancer therapies. We have therefore investigated expression changes in AXL and GAS6 in 15 AML cell lines and CD34+ stem/progenitor cells obtained from 11 primary AML patient samples. Interestingly, the transcript levels of AXL and GAS6 were found to be significantly increased in several AML cell lines carrying MLL fusion genes as compared to cells without MLL fusions (~6-fold and 8-fold). This result was further confirmed in these cells by Western blot analysis. Moreover, AXL transcripts were highly increased in primary CD34+ stem/progenitor cells as compared to more mature CD34- cells from nine AML patient samples studied (~7-fold). Transcript levels of GAS6 were slightly increased in CD34+ cells compared to CD34- cells from the same patient samples. These results indicate that both AXL and GAS6 are highly expressed in AML cell lines harboring MLL fusion genes, as well as in patient stem/progenitor cells.Interestingly, we have further demonstrated that AML cells with high expression of GAS6/AXL were more sensitive to the highly selective and novel AXL inhibitor SLC-0211, as compared to AML cells with low expression of GAS6 and/or AXL, as assessed by viability and apoptosis assays (2-3 fold). Notably, AML cells with high expression of both GAS6 and AXL, but without FLT3-ITD mutation, were highly sensitive to SLC-0211 treatment compared to other AXL inhibitors (>10-fold). Additive effects of SLC-0211 with chemotherapeutic drugs cytarabine or daunorubicin were also observed. In addition, SLC-0211 modestly reduced the colony forming ability of CD34+ AML cells, but the inhibitory effect of SLC-0211 was strikingly enhanced in re-plating assays, resulting in a significant reduction in re-plating efficiency of more primitive AML cells (75-97% inhibition). Importantly, the drug concentrations used to inhibit AML CFCs were not toxic to normal human CD34+ stem/progenitor cells. At the molecular level, treatment with SLC-0211 greatly reduced phosphorylation of AXLY779, AKTS473 and ERKT202/Y204 in AML cells in a dose-dependent manner as compared to control cells, as demonstrated by Western blot analysis. Taken together, these results indicate that inhibition of AXL by SLC-0211 specifically targets primitive AML cells where the AXL/GAS6 pathway is highly activated. Thus, targeting AXL may provide improved therapies for specific subgroups of AML. DisclosuresNo relevant conflicts of interest to declare.

AbstractThe abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

Carbonic anhydrase IX (CAIX) is considered a target for therapeutic intervention in solid tumors. In this study, the efficacy of the inhibitor, 4-(3-(2,4-difluorophenyl)-oxoimidazolidin-1-yl)benzenesulfonamide (SLC-149), is evaluated on CAIX and a CAIX-mimic. We show that SLC-149 is a better inhibitor than acetazolamide against CAIX. Binding of SLC-149 thermally stabilizes CAIX-mimic at lower concentrations compared to that of CAII. Structural examinations of SLC-149 bound to CAIX-mimic and CAII explain binding preferences. In cell culture, SLC-149 is a more effective inhibitor of CAIX activity in a triple-negative breast cancer cell line than previously studied sulfonamide inhibitors. SLC-149 is also a better inhibitor of activity in cells expressing CAIX versus CAXII. However, SLC-149 has little effect on cytotoxicity, and high concentrations are required to inhibit cell growth, migration, and invasion. These data support the hypothesis that CAIX activity, shown to be important in regulating extracellular pH, does not underlie its ability to control cell growth.

Acute myeloid leukemia (AML) is a heterogeneous haematological cancer characterized phenotypically by the rapid clonal growth of myeloid cells and an accumulation of blasts in the peripheral blood and bone marrow of patients. Despite the major progress that has been made in categorizing different genetic and molecular AML subgroups, therapies and long-term patient outcomes have not changed significantly over the past four decades. Recently, venetoclax (ABT-199), a BH3-mimetic and selective BCL-2 inhibitor, was approved for the treatment of older patients with AML. However, the limited efficacy, drug resistance in complex karyotype AML and disease progression on venetoclax as well as the inherent resistance of leukemic stem cells (LSCs) to therapy pose significant clinical challenges, warranting identification of novel targets and improved treatment strategies. One candidate target is AXL, a member of the TYRO3/AXL/MER (TAM) family of receptor tyrosine kinases. AXL and its ligand growth arrest-specific gene 6 (GAS6) are elevated in AML patients and LSCs, and associated with poor prognosis. To test whether targeting of the AXL/GAS6 pathway is a feasible treatment strategy for AML, in particular to eradicate LSCs, we developed SLC-391, a novel, potent and selective AXL inhibitor. In vitro and in vivo evaluations of the pharmaceutical properties of SLC-391 indicated reasonable solubility, excellent metabolic stability as well as desirable bioavailability in mice and rats. In silico molecular docking analysis showed that SLC391 can adopt a conformation with surface and charge complementary to the active site of the AXL kinase, potentially engaging in hydrophobic ring-mediated interactions. Further, cell-based studies discovered that SLC-391 targets AML cells with high AXL/GAS6 expression, particularly MLL+ AML cells, and synergizes with venetoclax in cell viability and apoptosis assays (CI<0.6). In addition, simultaneous AXL and BCL-2 inhibition reduced the clonal short- and long-term growth of primitive AML patient cells in CFC re-plating and LTC-IC assays compared to single or control treatments (20-95% inhibition). Moreover, a combination of AXL inhibition and venetoclax treatment was able to target LSCs and AML blasts in two different preclinical patient-derived xenotransplantation (PDX) models, extending the mean survival of these mice by 14-30 days compared to single agents (P<0.025). Mechanistically, single-cell RNA-sequencing and functional validation studies revealed that AXL inhibition perturbs oxidative metabolism, and differentially targets signaling pathways to synergize with venetoclax in leukemic cell killing. Importantly, the combination of AXL inhibition plus venetoclax treatment was not toxic to normal BM cells from healthy donors. Hence, our findings identify a promising, improved and specific treatment strategy for AML, particularly patients with high AXL/GAS6 expression. Disclosures No relevant conflicts of interest to declare.

Abstract Carbonic anhydrase IX (CAIX) is a cell surface, HIF-1A-inducible enzyme that is highly expressed in many solid tumors, is considered to be an endogenous marker of hypoxia and is a prominent biomarker of poor patient prognosis. In contrast, CAIX expression in normal human normal tissue is highly restricted, making it an attractive therapeutic target and driving the development of CAIX-specific inhibitors. SLC-0111 is an ureido-substituted benzenesulfonamide small molecule inhibitor of CAIX. We have shown previously that targeting CAIX activity with SLC-0111, alone and in combination with chemotherapy or immune checkpoint blockade, results in anti-tumor efficacy in multiple solid tumor models in vivo. Here, we report the findings of a multi-center, open-label, first-in-human phase I clinical trial investigating the safety and tolerability of SLC-0111 in patients with previously treated, advanced solid tumors (NCT02215850). Using a 3+3 design, dose escalation started at 500 mg oral daily dosing of SLC-0111 in cohort 1 and increased to 1000 mg and 2000 mg in cohorts 2 and 3. Drug-related adverse events (AEs) were monitored to determine safety and tolerability. Pharmacokinetic analyses assessed plasma concentrations of single and repeated doses of SLC-0111. RECIST 1.1 criteria were used to assess disease progression. No DLTs were reported and patients dosed at 1000 mg and below exhibited fewer drug-related AEs ≥ grade 3 and fewer AEs such as nausea and vomiting, compared to the 2000 mg cohort. 41% of patients experienced dose interruptions or discontinuation and the majority (71%) of these occurred in the 2000 mg cohort. Mean Cmax and AUC(0-24) values for single doses were similar at the 1000 mg and 2000 mg dose levels. Mean Tmax and T1/2 values of SLC-0111 were similar after single and repeated dosing. Power-law analysis of Cmax and AUC(0-24) showed that exposure to SLC-0111 was generally dose proportional. No objective responses were observed, but stable disease greater than 24 weeks was observed in 2 patients. These data show that SLC-0111 is safe in patients and support 1000 mg/day as the recommended phase II dose (RP2D). Based on these encouraging results, a phase Ib clinical trial to evaluate SLC-0111 in combination with chemotherapy in a defined population of CAIX-positive pancreatic cancer patients (NCT03450018) is now open and future clinical development will include investigation of SLC-0111 in combination with chemotherapy in patients with glioblastoma. Citation Format: Paul C. McDonald, Stephen Chia, Philippe L. Bedard, Qunicy Chu, Michael Lyle, Liren Tang, Madhu Singh, Zaihui Zhang, Claudiu T. Supuran, Daniel J. Renouf, Shoukat Dedhar. Phase I clinical trials of SLC-0111, a novel inhibitor of carbonic anhydrase IX, in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT161.

Acute myeloid leukemia (AML) is an aggressive haematological cancer characterized phenotypically by the rapid clonal growth of myeloid cells and an accumulation of immature blasts. Although major progress has been made in identifying different genetic and molecular AML subgroups, treatment and long-term patient outcomes have not changed significantly over the past four decades. Recently, venetoclax (ABT-199), a selective BCL-2 inhibitor, has been approved for the treatment of older patients with AML. However, drug resistance and disease progression on venetoclax as well as the inherent resistance of leukemic stem cells (LSCs) to therapy pose significant clinical challenges. One candidate target for improved therapies is AXL, a member of the TYRO3/AXL/MER (TAM) family of receptor tyrosine kinases that is elevated in AML cells and LSCs, and associated with poor prognosis in AML. To test whether targeting of AXL is a feasible treatment strategy for AML, in particular to eradicate LSCs, we developed a potent and selective AXL inhibitor. We show that AXL inhibition targets AML cells with high AXL expression and synergizes with venetoclax decreasing the viability of primitive AML patient cells and reducing their clonal short- and long-term colony assay outputs compared to single or control treatments. Moreover, a combination of AXL inhibition and venetoclax treatment was able to target LSCs and AML blasts in two different preclinical patient-derived xenotransplantation (PDX) models. Single-cell RNA-sequencing and functional validation studies revealed that AXL inhibition perturbs oxidative metabolism and differentially targets signaling pathways to synergize with venetoclax in leukemic cell killing. Importantly, the combination of AXL inhibition plus venetoclax was not toxic to normal BM cells from healthy donors. Hence, our findings identify a promising, improved and specific treatment strategy for AML.

SLC-0111 is an ureido-substituted benzenesulfonamide small molecule inhibitor of carbonic anhydrase IX. The objectives of this first-in-human Phase 1 study were to determine the safety and tolerability of SLC-0111 in patients with advanced solid tumors and to establish the recommended Phase 2 dose for future clinical investigations. Using a 3+3 design, dose escalation started at 500 mg oral daily dosing of SLC-0111 in cohort 1 and increased to 1000 and 2000 mg in cohorts 2 and 3. Drug-related adverse events (AEs) were monitored to determine safety and tolerability. Pharmacokinetic analyses assessed plasma concentrations of single and repeated doses of SLC-0111. RECIST 1.1 criteria were used to assess disease progression. No dose-limiting toxicities were reported and patients dosed at ≤1000 mg exhibited fewer drug-related AEs ≥ grade 3 and fewer AEs such as nausea and vomiting, compared with the 2000-mg cohort. Forty-one percent of patients experienced dose interruptions or discontinuation and the majority (71%) of these occurred in the 2000-mg cohort. Mean Cmax and AUC(0-24) values for single doses were similar at the 1000-mg and 2000-mg dose levels. Mean Tmax and T1/2 values of SLC-0111 were similar after single and repeated dosing. Power-law analysis of Cmax and AUC0-24 showed that exposure to SLC-0111 was generally dose proportional. No objective responses were observed, but stable disease >24 weeks was observed in 2 patients. SLC-0111 was safe in patients with previously treated, advanced solid tumors. The safety and pharmacokinetic data support 1000 mg/d as the recommended phase 2 dose for SLC-0111.

Isosteric replacement of amide groups is a classic practice in medicinal chemistry. This digest highlights the applications of most commonly employed amide isosteres in drug design aiming at improving potency and selectivity, optimizing physicochemical and pharmacokinetic properties, eliminating or modifying toxicophores, as well as providing novel intellectual property of lead compounds.