Generative artificial intelligence (AI) is now widely applied in medicinal chemistry, with detailed case studies emerging in the literature. Here, we describe an early application of REINVENT, AstraZeneca's in-house generative molecular design platform, to identify new inhibitor scaffolds for hematopoietic progenitor kinase 1 (HPK1). REINVENT was deployed at two stages of the project to address distinct design objectives. For hit identification, transfer learning on kinase-active compounds, followed by reinforcement learning guided by QSAR-based scoring, led to the discovery of three active chemotypes. Subsequently, REINVENT was applied to scaffold hopping, using 3D pharmacophore and docking models as scoring functions, which enabled the identification of two additional active chemotypes. Optimization of one of these scaffolds delivered a compound with potent cellular activity, kinase selectivity, and favorable rat pharmacokinetics. These results demonstrate the value of integrating generative AI with medicinal chemistry expertise and support broader application of the approach in future discovery programs.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of the T cell receptor signaling pathway and is therefore a target of interest for immunooncology. Nonselective HPK1 inhibitors may affect other kinase components of T cell activation, blunting the beneficial impact of enhanced T cell activity that results from HPK1 inhibition itself. Here, we report the discovery of pyrazine carboxamide HPK1 inhibitors and their optimization through structure-based drug design to afford a highly selective HPK1 inhibitor, compound 24 (AZ3246). This compound induces IL-2 secretion in T cells with an EC50 of 90 nM without inhibiting antagonistic kinases, exhibits pharmacokinetic properties consistent with oral dosing, and demonstrates antitumor activity in the EMT6 syngeneic mouse model.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

We report here a fragment screen directed toward the c-MET kinase from which we discovered a series of inhibitors able to bind to a rare conformation of the protein in which the P-loop adopts a collapsed, or folded, arrangement. Preliminary SAR exploration led to an inhibitor (7) with nanomolar biochemical activity against c-MET and promising cell activity and kinase selectivity. These findings increase our structural understanding of the folded P-loop conformation of c-MET and provide a sound structural and chemical basis for further investigation of this underexplored yet potentially therapeutically exploitable conformational state.

Abstract Introduction: Loss of immune surveillance is required for cancer cell growth and metastasis; tumors co-opt suppressive mechanisms to evade detection by the immune system. The immune system is equipped with multiple feedback mechanisms to limit inflammation to prevent autoimmunity; tumors activate these pathways to escape immunity. HPK1 (hematopoietic progenitor kinase 1) is a negative regulator of T-cell activation; the kinase activity limits T-cell signaling and tumoricidal cytokine production. Recent literature has shown that inactivation of the kinase function of HPK1 prevents tumor progression in murine tumor models (1). Experimental Procedures: We used lentiviral delivered shRNAs and CRISPR/Cas9 technology to delete HPK1 in Jurkat cells and primary human T cells, respectively. Jurkat T cells (wt/KO) were activated with anti-CD3 antibody and cell lysates were assessed by Western blot to examine signaling events downstream of the T-cell receptor (TCR). Primary human T cells or CRISPR/Cas9 KO T cells were activated in vitro using anti-CD3/anti-CD28 antibodies in the presence or absence of prostaglandin E2 (PGE2). Cytokines were quantified by ELISA. Results: We present novel findings demonstrating that the role of HPK1 is conserved in human Jurkat cells as well as in primary human T cells. Loss of HPK1 enhanced T-cell receptor signaling in Jurkat cells. In CRISPR/Cas9 KO HPK1 primary human T cells, TCR activation resulted in enhanced cytokine secretion and proliferation concomitant to a decrease in pSLP76, the target molecule phosphorylated by HPK1. In addition, HPK1 KO cells were rescued from PGE2-mediated suppression. A screening and assessment of HPK-1 inhibitor compounds that enhance T-cell function is ongoing. Conclusions: In summary, HPK1 function is conserved in human T cells and inhibitors of HPK1 could enhance antitumor immunity through increased T-cell function, overcoming suppressive signals in the tumor microenvironment and broadening the response to check point inhibitors for cancer immunotherapy. Reference 1. Hernandez S, Qing J et al. The kinase activity of hematopoietic progenitor kinase 1 is essential for the regulation of T cell function. Cell Rep 2018;25:80-94. Citation Format: Neil Grimster, Lisa Drew, Stephen Fawell, Deanna A Mele, Gayathri Bommakanti, Minhui Shen, Kevin Xu, Kun Song, Rob Ziegler, Jason Kettle, Adelphe Mfuh, Jason Sheilds. Releasing the brake on T-cell activation through inhibition of HPK1 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A56.

Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and correct maladaptive mineral and hormonal derangements associated with increased cardiovascular risk in chronic kidney disease-mineral and bone disorder (CKD-MBD). To date, only nonselective NaPi inhibitors have been described. Herein, we detail the discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic β 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22 was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na+/K+ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it could be suitable as a potential clinical asset.

Inducing α-helicity through side-chain cross-linking is a strategy that has been pursued to improve peptide conformational rigidity and bio-availability. Here we describe the preparation of small peptides tethered to chiral sulfoxide-containing macrocyclic rings. Furthermore, a study of structure-activity relationships (SARs) disclosed properties with respect to ring size, sulfur position, oxidation state, and stereochemistry that show a propensity to induce α-helicity. Supporting data include circular dichroism spectroscopy (CD), NMR spectroscopy, and a single crystal X-ray structure for one such stabilized peptide. Finally, theoretical studies are presented to elucidate the effect of chiral sulfoxides in inducing backbone α-helicity.

The Na+/citrate transporter, NaCT (SLC13A5), is a therapeutic target for metabolic diseases. Citrate is an important signaling molecule that regulates the activity of lipid- and glucose-metabolizing enzymes in cells. Previous studies identified two compounds, PF-06649298 (compound 2: ) and PF-06678419 (compound 4: ), that inhibit human NaCT with high affinity, and one of the compounds demonstrated specificity relative to other SLC13 family members. Here we use molecular modeling and site-directed mutagenesis of hNaCT followed by transport characterization and cell-surface biotinylation to examine the residues involved in inhibitor binding and transport. The results indicate that residues located near the putative citrate binding site, G228, V231, V232, and G409, affect both citrate transport and inhibition of citrate uptake by compounds 2: and 4: V231 appears to distinguish between compounds 2: and 4: as inhibitors. Furthermore, residues located outside of the putative citrate binding site, Q77 and T86, may also play a role in NaCT inhibition by compounds 2: and 4: Our results provide new insight into the mechanism of transport and inhibition in NaCT and the SLC13 family. These findings should provide a basis for future drug design of SLC13 inhibitors.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

A novel series of nonsteroidal mineralocorticoid receptor (MR) antagonists identified as part of our strategy to follow up on the clinical candidate PF-03882845 (2) is reported. Optimization departed from the previously described pyrazoline 3a and focused on improving the selectivity for MR versus the progesterone receptor (PR) as an approach to avoid potential sex-hormone-related adverse effects and improving biopharmaceutical properties. From this effort, (R)-14c was identified as a potent nonsteroidal MR antagonist (IC50 = 4.5 nM) with higher than 500-fold selectivity versus PR and other related nuclear hormone receptors, with improved solubility as compared to 2 and pharmacokinetic properties suitable for oral administration. (R)-14c was evaluated in vivo using the increase of urinary Na(+)/K(+) ratio in rat as a mechanism biomarker of MR antagonism. Treatment with (R)-14c by oral administration resulted in significant increases in urinary Na(+)/K(+) ratio and demonstrated this novel compound acts as an MR antagonist.

A general method for constraining peptide conformations via linkage of aromatic sidechains has been developed. Macrocyclization of suitably functionalized tri-, tetra- and pentapeptides via Suzuki-Miyaura cross-coupling has been used to generate side chain to side chain, biaryl-bridged 14- to 21-membered macrocyclic peptides. Biaryl bridges possessing three different configurations, meta-meta, meta-ortho, and ortho-meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki-Miyaura macrocyclizations were successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.