Ion channel selectivity through ion-modulated changes of selectivity filter pKa values.

In bacterial voltage-gated sodium channels, the passage of ions through the pore is controlled by a selectivity filter (SF) composed of four glutamate residues. The mechanism of selectivity has been the subject of intense research, with suggested mechanisms based on steric effects, and ion-triggered conformational change. Here, we propose an alternative mechanism based on ion-triggered shifts in pKa values of SF glutamates. We study the NavMs channel for which the open channel structure is available. Our free-energy calculations based on molecular dynamics simulations suggest that pKa values of the four glutamates are higher in solution of K+ ions than in solution of Na+ ions. Higher pKa in the presence of K+ stems primarily from the higher population of dunked conformations of the protonated Glu sidechain, which exhibit a higher pKa shift. Since pKa values are close to the physiological pH, this results in predominant population of the fully deprotonated state of glutamates in Na+ solution, while protonated states are predominantly populated in K+ solution. Through molecular dynamics simulations we calculate that the deprotonated state is the most conductive, the singly protonated state is less conductive, and the doubly protonated state has significantly reduced conductance. Thus, we propose that a significant component of selectivity is achieved through ion-triggered shifts in the protonation state, which favors more conductive states for Na+ ions and less conductive states for K+ ions. This mechanism also suggests a strong pH dependence of selectivity, which has been experimentally observed in structurally similar NaChBac channels.

Determinants of conductance of a bacterial voltage-gated sodium channel.

Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel NavMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Nav channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na+ in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.

Protonation state of the selectivity filter of bacterial voltage-gated sodium channels is modulated by ions.

The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C4 symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Nav Ms) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C4 symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C2 symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Optimization beyond AMG 232: discovery and SAR of sulfonamides on a piperidinone scaffold as potent inhibitors of the MDM2-p53 protein-protein interaction.

We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.

Benefits and barriers to return to education and relationship to quality of life for people living with spinal cord injury: Results of a mixed methods study.

CONTEXT: There is limited research on return to education (RTE) after spinal cord injury (SCI). As a result, few programs exist to help people achieve this goal. OBJECTIVE: The primary objective was to investigate the barriers and facilitators to RTE, and the relationship between RTE and quality of life (QOL). The secondary objective was to examine the role of a Vocational Resource Facilitation (VRF) program on RTE. METHODS: A mixed methods approach with a semi-structured interview and online survey was used. Participants included 15 people with SCI with RTE goals who received VRF services at an acute inpatient rehabilitation hospital. Qualitative responses on the barriers, facilitators and perceived benefits of RTE were analyzed using a grounded theory strategy. RESULTS: Barriers to RTE included physical and mental health, transportation, time, environmental barriers, finances, lack of knowledge about available resources, and discrimination. Facilitators to RTE included the VRF program, social support, financial support, virtual learning, organizational support, and policy constructs. People who RTE after SCI reported better QOL, less depressed mood than those who did not, and were more likely to have returned to work. CONCLUSIONS: This study highlighted common barriers and facilitators to RTE, and potential areas of intervention. VRF is a potentially efficacious early intervention vocational rehabilitation approach that improves participation in education and employment for people with SCI. People who received VRF and achieved RTE may have better QOL outcomes and improved employability.

Characterization of AB598, a CD39 Enzymatic Inhibitory Antibody for the Treatment of Solid Tumors.

AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular ATP (eATP), an alarmin capable of promoting anti-tumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a pro-inflammatory milieu in which eATP can activate myeloid cells to promote anti-tumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemically dosed AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive anti-tumor immune response.

Protein pKa Prediction by Tree-Based Machine Learning.

Protonation states of ionizable protein residues modulate many essential biological processes. For correct modeling and understanding of these processes, it is crucial to accurately determine their pKa values. Here, we present four tree-based machine learning models for protein pKa prediction. The four models, Random Forest, Extra Trees, eXtreme Gradient Boosting (XGBoost), and Light Gradient Boosting Machine (LightGBM), were trained on three experimental PDB and pKa datasets, two of which included a notable portion of internal residues. We observed similar performance among the four machine learning algorithms. The best model trained on the largest dataset performs 37% better than the widely used empirical pKa prediction tool PROPKA and 15% better than the published result from the pKa prediction method DelPhiPKa. The overall root-mean-square error (RMSE) for this model is 0.69, with surface and buried RMSE values being 0.56 and 0.78, respectively, considering six residue types (Asp, Glu, His, Lys, Cys, and Tyr), and 0.63 when considering Asp, Glu, His, and Lys only. We provide pKa predictions for proteins in human proteome from the AlphaFold Protein Structure Database and observed that 1% of Asp/Glu/Lys residues have highly shifted pKa values close to the physiological pH.

Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.

Design, Synthesis, and Structure-Activity Relationship Optimization of Pyrazolopyrimidine Amide Inhibitors of Phosphoinositide 3-Kinase γ (PI3Kγ).

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,ß-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Discovery of Potent and Selective 7-Azaindole Isoindolinone-Based PI3Kγ Inhibitors.

The successful application of immunotherapy in the treatment of cancer relies on effective engagement of immune cells in the tumor microenvironment. Phosphoinositide 3-kinase γ (PI3Kγ) is highly expressed in tumor-associated macrophages, and its expression levels are associated with tumor immunosuppression and growth. Selective inhibition of PI3Kγ offers a promising strategy in immuno-oncology, which has led to the development of numerous potent PI3Kγ inhibitors with variable selectivity profiles. To facilitate further investigation of the therapeutic potential of PI3Kγ inhibition, we required a potent and PI3Kγ-selective tool compound with sufficient metabolic stability for use in future in vivo studies. Herein, we describe some of our efforts to realize this goal through the systematic study of SARs within a series of 7-azaindole-based PI3Kγ inhibitors. The large volume of data generated from this study helped guide our subsequent lead optimization efforts and will inform further development of PI3Kγ-selective inhibitors for use in immunomodulation.

Discovery of Potent and Selective PI3Kγ Inhibitors.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 µM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Discovery of Potent and Selective Non-Nucleotide Small Molecule Inhibitors of CD73.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

Discovery of AB680: A Potent and Selective Inhibitor of CD73.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (Ki = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-Inducing Cytotoxic Agents.

p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2-p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics.

Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses.

Discovery of AM-7209, a potent and selective 4-amidobenzoic acid inhibitor of the MDM2-p53 interaction.

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Novel inhibitors of the MDM2-p53 interaction featuring hydrogen bond acceptors as carboxylic acid isosteres.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.