Juan Alvarez

Pharmacophore‐based molecular docking to account for ligand flexibility

Rapid computational mining of large 3D molecular databases is central to generating new drug leads. Accurate virtual screening of large 3D molecular databases requires consideration of the conformational flexibility of the ligand molecules. Ligand flexibility can be included without prohibitively increasing the search time by docking ensembles of precomputed conformers from a conformationally expanded database. A pharmacophore‐based docking method whereby conformers of the same or different molecules are overlaid by their largest 3D pharmacophore and simultaneously docked by partial matches to that pharmacophore is presented. The method is implemented in DOCK 4.0. Proteins 2003;51:172–188.

Automated generation of MCSS‐derived pharmacophoric DOCK site points for searching multiconformation databases

All docking methods employ some sort of heuristic to orient the ligand molecules into the binding site of the target structure. An automated method, MCSS2SPTS, for generating chemically labeled site points for docking is presented. MCSS2SPTS employs the program Multiple Copy Simultaneous Search (MCSS) to determine target‐based theoretical pharmacophores. More specifically, chemically labeled site points are automatically extracted from selected low‐energy functional‐group minima and clustered together. These pharmacophoric site points can then be directly matched to the pharmacophoric features of database molecules with the use of either DOCK or PhDOCK to place the small molecules into the binding site. Several examples of the ability of MCSS2SPTS to reproduce the three‐dimensional pharmacophoric features of ligands from known ligand–protein complex structures are discussed. In addition, a site‐point set calculated for one human immunodeficiency virus 1 (HIV1) protease structure is used with PhDOCK to dock a set of HIV1 protease ligands; the docked poses are compared to the corresponding complex structures of the ligands. Finally, the use of an MCSS2SPTS‐derived site‐point set for acyl carrier protein synthase is compared to the use of atomic positions from a bound ligand as site points for a large‐scale DOCK search. In general, MCSS2SPTS‐generated site points focus the search on the more relevant areas and thereby allow for more effective sampling of the target site. Proteins 2003;51:189–202.

Critical evaluation of methods to incorporate entropy loss upon binding in high‐throughput docking

Proper accounting of the positional/orientational/conformational entropy loss associated with protein–ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics‐based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function‐based scoring (PFS) method, to account for such entropy losses in high‐throughput docking calculations. Our results on the estrogen receptor β and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over‐penalizes molecules for their conformational flexibility, the PFS method behaves in a more “ΔG‐like” manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead‐like random molecular database suggest that an accurate representation of the “true” energy landscape of the protein–ligand complex is critical for reliable predictions of relative binding affinities by the PFS method. Proteins 2007.

Design and synthesis of sialyl Lewis x mimics as E-selectin inhibitors.

The design and synthesis of novel beta-C-mannosides that inhibit the binding of sialyl Lewis x to E-selectin are described. Compounds that contained a phenyl substituent at the C-6 position were found to have increased potency.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 9: Synthesis, characterization and molecular modeling of pyridinyl isosteres of N-BPE-8-CAC (1), a high affinity ligand for opioid receptors.

Derivatives of the lead compound N-BPE-8-CAC (1) where each CH of the biphenyl group was individually replaced by N were prepared in hopes of identifying high affinity ligands with improved aqueous solubility. Compared to 1, binding affinities of the five possible pyridinyl derivatives for the μ opioid receptor were between threefold lower to fivefold higher with the Ki of the most potent compound being 0.064 nM. Docking of 8-CAC (2) into the unliganded binding site of the mouse μ opioid receptor (pdb: 4DKL) revealed that 8-CAC and β-FNA (from 4DKL) make nearly identical interactions with the receptor. However, for 1 and the new pyridinyl derivatives 4-8, binding is not tolerated in the 8-CAC binding mode due to the steric constraints of the large N-substituents. Either an alternative binding mode or rearrangement of the protein to accommodate these modifications may account for their high binding affinity.

Abstract 4088: First-in-human dose selection for ALKS 4230, an investigational immunotherapeutic agent

ALKS 4230 is an engineered fusion protein comprised of a circularly permuted interleukin-2 (IL-2) and the IL-2 receptor (IL-2R) α chain, CD25, designed to selectively activate the intermediate-affinity IL-2R, but not the high-affinity IL-2R. Selective activation of the intermediate-affinity IL-2R by ALKS 4230 has the potential to provide enhanced tumor killing as well as improved safety and tolerability. Various in vitro and in vivo studies were conducted to characterize the primary and secondary pharmacodynamics (PD) of ALKS 4230 as well as its pharmacokinetics (PK). The results guided the selection of the starting dose for the ALKS 4230 first-in-human (FIH) clinical study based upon the Minimal Anticipated Biological Effect Level (MABEL) approach. The PK-PD relationship for ALKS 4230 was evaluated in in vitro pharmacology studies in target cells from murine, non-human primate and human donors. The mean EC10 values for activation of NK cells, memory CD8 T-cells and Tregs in target cells from human donors were 0.09, 0.18 and 0.13 nM, respectively. Using the lowest EC10 value of 0.09 nM (0.0031µg/mL) as a surrogate for the MABEL, assuming IV administration to a 70 kg human with 3 L plasma volume, a dose of 0.1 µg/kg would be expected to result in an immediate post-dose concentration of 0.0031 µg/mL. The mean EC50 values for activation of NK cells, memory CD8 T cells and Tregs in target cells from human donors were 0.46, 1.1 and 0.59 nM, respectively. Using the EC50 value as surrogate for minimal effective ALKS 4230 concentration that induces activation of human IL-2R complex, the projected minimal efficacious dose (MED) in humans to achieve a concentration of 0.46 nM (0.016 µg/mL) to 1.1 nM (0.038 µg/mL) is 0.7- 1.6 µg/kg. Based on a MABEL dose of 0.1 µg/kg and projected MED of 0.7- 1.6 µg/kg, the proposed doses to be evaluated in the FIH Phase 1 study are 0.1, 0.3, 1, 3, 10, and 30 µg/kg. In comparison, the projected Cmax at the proposed starting dose of 0.1 µg/kg is > 750-fold lower than the Cmax at the no-observed adverse effect level (NOAEL) in a repeat-dose toxicology study in monkeys. It is also about 3-fold lower than the lowest concentration of ALKS 4230 (0.01 µg/mL) tested in the cytokine release assays at which only slight elevations were observed for IL-6, IL-8, and IFN-γ in a small number of whole blood samples from healthy human donors, similar to those in the low-response control across the concentration range evaluated. Therefore, 0.1 µg/kg is considered a safe starting dose for the FIH study. The PK, PD and toxicology assessments conducted to date support the FIH investigation of ALKS 4230 at the proposed starting dose of 0.1 µg/kg. Citation Format: Lei Sun, Heather C. Losey, Juan Alvarez, Lisa von Moltke, William J. Slichenmyer. First-in-human dose selection for ALKS 4230, an investigational immunotherapeutic agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4088. doi:10.1158/1538-7445.AM2017-4088

Abstract 3158: Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells

RDB 1450 is an engineered fusion protein of circularly permuted IL-2 and IL-2Rα that is selective for the intermediate-affinity IL-2 receptor. The intermediate-affinity receptor is comprised of the IL-2 receptor beta chain (CD122) and common gamma chain (CD132) expressed on lymphocytes with effector function, whereas the high-affinity receptor, which additionally includes the alpha chain (CD25), is expressed primarily on immunosuppressive CD4+CD25+ regulatory T cells (Tregs). Ex vivo proliferation of subsets of human lymphocytes in response to RDB 1450 or rhIL-2 were evaluated. RDB 1450 supports the selective activation and expansion of effector lymphocytes based on higher observed memory CD8 T/Treg and NK/Treg ratios. Both RDB 1450 and IL-2 drive expansion of NK cells, however, a greater proportion of CD25+ NK cells are observed in response to RDB 1450. Furthermore, unlike IL-2, RDB 1450 does not effectively expand highly suppressive ICOS+ Tregs. The immunological profile resulting from the ex vivo treatment of human PBMCs with RDB 1450 supports its use as a novel immunotherapy for cancer. Citation Format: Jared E. Lopes, Jan L. Fisher, Heather C. Losey, Marc S. Ernstoff, Juan C. Alvarez. Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3158. doi:10.1158/1538-7445.AM2015-3158