Accurately measuring compound binding affinities is key to driving the pharmaceutical development process. Rigorous physics-based in silico approaches, particularly alchemical free energy methods, have become a gold-standard tool for estimating compound affinity changes. Here we present the results of a large-scale precompetitive collaborative assessment of relative binding free energy (RBFE) calculations generated by 15 pharmaceutical companies. We evaluate an open-source and MIT-licensed RBFE protocol from the Open Free Energy (OpenFE) ecosystem across both public and blinded private datasets, encompassing over 1,700 ligands in total. For the public dataset, the weighted RMSE across the 58 systems was 1.73(1.53)(1.96) kcal/mol, with 10 of the systems reaching sub-kcal/mol accuracy. For the private dataset, the weighted RMSE across the 37 systems was 2.44(1.94)(3.06) kcal/mol, with only 2 of the systems reaching sub-kcal/mol accuracy, reflecting the increased complexity of real-world drug discovery. The protocol's performance was system-dependent, with no single dominant error source, indicating that accuracy is primarily influenced by input quality and transformation type. Overall, these benchmark results are encouraging and indicate that OpenFE is ready for large-scale industrial applications, with an "out-of-the-box" accuracy that approaches that of commercial solutions. While comparison against published FEP+ results, which were obtained after manual parameter optimization, shows comparable ranking statistics, a gap remains in error statistics, for which we outline possible paths toward improvement. The protocol meets key criteria required for production use in an industrial setting: it shows robust performance, generates reproducible results, and achieves both sufficient throughput and rapid convergence.

Mutant KRAS is highly prevalent in human cancer and has been actively pursued as a target for drug discovery. Much progress has been made in drugging KRAS G12C, owing to the ability of inhibitors to covalently target its oncogenic cysteine mutation at codon 12. A number of KRAS G12C inhibitors have advanced to clinical development and are being investigated for the treatment of a variety of solid tumors. Notably, many patients with KRAS G12C-positive non-small cell lung cancer develop brain metastases. Herein, we report the discovery and development of a brain-penetrant inhibitor of KRAS G12C using divarasib as a starting point. Optimization efforts focused on reducing molecular weight and topological polar surface area as well as shielding of hydrogen bond donors. In this manner, active transport by both P-gp and breast cancer resistance protein (BCRP) was attenuated, and high exposure in rodent brain tissue was achieved.

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.

Oncogenic KRAS mutations were identified decades ago, yet the selective inhibition of specific KRAS mutant proteins represents an ongoing challenge. Recent progress has been made in targeting certain P-loop mutant proteins, in particular KRAS G12C, for which the covalent inhibition of the GDP state via the Switch II pocket is now a clinically validated strategy. Inhibition of other KRAS mutant proteins such as KRAS G13D, on the other hand, still requires clinical validation. The remoteness of the D13 residue relative to the Switch II pocket in combination with the solvent exposure and conformational flexibility of the D13 side chain, as well as the difficulties of targeting carboxylate residues covalently, renders this specific protein particularly challenging to target selectively. In this report, we describe the design and evaluation of potent and KRAS G13D-selective reversible inhibitors. Subnanomolar binding to the GDP state Switch II pocket and biochemical selectivity over WT KRAS are achieved by leveraging a salt bridge with D13.

Background: Many HER2-directed antibody-drug conjugates (ADCs) in early development employ trastuzumab linked to tubulin binding agents as the cytotoxic drug. DHES0815A is an ADC consisting of a THIOMABTM humanized IgG1 anti-HER2 monoclonal antibody (hu7C2), which binds domain 1 of the HER2 ECD, conjugated via a disulfide linker to PBD-monoamide (MA), a DNA mono-alkylating agent. The reduced potency of the PBD-MA payload compared to PBD dimers and the stability of the conjugation site and linker were designed to improve tolerability, whereas the binding of hu7C2 to a HER2 epitope distinct from trastuzumab and pertuzumab allows combination with existing HER2 therapies. . Methods: Modifications at one imine in the PBD (pyrrolobenzodiazepine) dimer produced mono-alkylating PBDs that were assessed for DNA binding, permeability and cell potency. Each modified PBD was conjugated to hu7C2 LC K149C via a disulfide linker and assessed in HER2-positive models in vitro and in vivo. Safety studies were performed in female and male cynomolgus monkey treated every 3 weeks X 5, with a 7-week follow-up. Assessments included hematology, clinical chemistry, anti-drug antibody, standard clinical, neurological and ophthalmic exams and histopathology. Results: Our goal was design of a reduced potency PBD dimer ADC to achieve dosing in the linear PK range for optimal exposure, efficacy and safety. PBD dimers contain 2 reactive imines that bis-alkylate and crosslink DNA. Mono-alkylating PBD dimers were assessed in DNA binding, potency and permeability assays to select the optimal mono-alkylator, PBD-MA, for further investigation. hu7C2-disulfide-PBD-MA, or DHES0815A, was potent across HER2-positive breast and gastric cancer models in vitro and in vivo, with little effect on HER2-negative tumors or normal primary cells in vitro. DHES0815A was more potent than T-DM1 in HER2-positive and HER2-low breast and gastric cancer PDX models and demonstrated combination activity with standard of care agents T-DM1 or docetaxel. Mechanistically, treatment of HER2-positive cells with DHES0815A resulted in late S-phase cell cycle arrest; induction of DNA damage markers γH2AX, phospho-p53, phospho-CHK2, induction of apoptosis, and bystander activity. Pharmacokinetic analysis showed dose-proportional PK, with linear biphasic PK >/= 4mg/kg in cynomolgus monkey. Safety studies in cynomolgus monkey treated with 4, 8 or 12 mg/kg demonstrated that findings were non-adverse, monitorable and manageable. All doses were tolerated with few clinical observations and minimal to mild clinical pathology changes. Target organs were skin, bone marrow, lung and eye. The HNSTD (highest non-severely toxic dose) was 12 mg/kg. This dose enabled a starting dose of 0.6 mg/kg for phase 1 testing. Conclusions: DHES0815A is a HER2-directed ADC comprised of a domain I binding antibody linked to a reduced potency PBD dimer, PBD-MA, via a reducible disulfide linker. The antibody does not interfere with trastuzumab or pertuzumab binding, allowing combination with existing HER2 therapies. The reduced potency payload allows dosing in the linear PK range, supporting our hypothesis. DHES0815A demonstrated dose-dependent anti-tumor efficacy in multiple HER2+ breast and gastric cancer models, and showed efficacy in HER2-low models, where T-DM1 was inactive. Enhanced anti-tumor activity was observed combining DHES0815A with T-DM1, trastuzumab/pertuzumab or docetaxel. Studies in cynomolgus monkey demonstrated a safety profile compatible with initiating a phase 1 clinical trial. The phase 1 data will be presented in a separate presentation. Citation Format: Gail Lewis Phillips, Guangmin Li, Jun Guo, Shang-Fan Yu, Genee Lee, Donglu Zhang, Peter Dragovich, Thomas Pillow, Binqing Wei, M. Violet Lee, Ola Saad, Shab Masih, Douglas Leipold, Lisa Crocker, Melissa Schutten. Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-33.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Abstract Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a novel HER2 ADC, we selected an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. We modified the PBD dimer to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrated in vivo efficacy in models of HER2-positive and HER2-low cancers and was well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study in patients with HER2-positive metastatic breast cancer showed early signs of anti-tumor activity with limited toxicity. However, delayed dermal, ocular and pulmonary toxicities developed. The delayed onset, as well as non-resolvable nature of the toxicities resulted in termination of the phase 1 trial.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via coopted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro AD ME properties and the ability to induce cytokine production in primary human T-cells.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody-drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.