P1 Pocket Research Articles

Comparative study of the diversity of amino acids on human leucocyte antigen class II molecules in patients with acquired aplastic anaemia.

Human leucocyte antigen (HLA) class II molecules are critically involved in the pathology of acquired aplastic anaemia (AA) by regulating the immune response and autoreactive T cell-mediated haematopoietic cell death. In the study, amino acid residue variation and molecular structure of HLA class II have been initially investigated in 96 patients with AA. The frequencies of residues 9 and 57 in pocket 9 (P9) in DQB1, and amino acid positions 9, 11, 13, 16, 26, 38, 67 and 71 in the P4, P6 and P9 pockets in DRB1 were more prevalent among AA patients. By applying a multivariate recursive approach, the DRβ-Gln-16 (OR = 3.003, 95% CI = 1.468-6.145, pc = 0.003), DRβ-Ala-71 (OR = 1.924, 95% CI = 1.233-3.002, pc = 0.004) in P4/P7 and DQβ-Asp-57 (OR = 3.483, 95% CI = 1.079-11.242, pc = 0.037) in P9, these critical residues were significantly discovered as risk amino acid residues on the onset of AA, as well as associated with PNH-type cells and pathological somatic or cytogenetic mutations. In silico structural model analysis showed that identified DRβ-Ala-71 and DQβ-Asp-57 within the antigen-binding groove interacting with a more variable antigenic segments, may impact the repertoire of peptides presented, influence the interface HLA-antigen-T-cell receptor β (TCR β). These findings provided light on the immunogenetic pathophysiology of AA aetiology and their potential impact on upcoming immunotherapies.

Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro, including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy.

Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Synthesis, design, and antiproliferative evaluation of 6-(N-Substituted-methyl)pyrazolo[3,4-d]pyrimidines as the potent anti-leukemia agents

Pyrazolopyrimidine derivatives, including pyrazolopyrimidines, 6-aminopyrazolopyrimidines, 6-[(formyloxy)methyl]pyrazolopyrimidines, 6-(hydroxymethyl)pyrazolopyrimidine, and 6-(aminomethyl)pyrazolopyrimidines have been successfully prepared and tested against NCI-H226, NPC-TW01, and Jurkat cancer cell lines. Among the tested pyrazolopyrimidine compounds, we found 6-aminopyrazolopyrimidines and 6-(aminomethyl)pyrazolopyrimidines with essential o-ClPh or p-ClPh substituted moieties on N-1 pyrazole ring exhibited the best IC50 inhibition activity for Jurkat cells. Furthermore, optimization of the SAR study on the C-6 position of pyrazolopyrimidine ring demonstrated that 6-(N-substituted-methyl)pyrazolopyrimidines 17b, 17d, and 19d possessed the significant IC50 inhibitory activity for the different leukemia cell lines, especially for Jurkat, K-562, and HL-60. On the other hand, further SAR inhibition and docking model studies revealed that compound 19d, which has a 3-(1H-imidazol-1-yl)propan-1-amino side-chain on the C-6 position, was able to form four hydrogen bonds with residues Ala226, Leu152, and Glu194 and specifically extended into the P1 pocket subsite with Aurora A, resulting in improved inhibitory activity almost similar to SNS-314. To explore the anti-cancer mechanism, compound 19d was measured by Western blot analysis in Jurkat T-cells, however, it showed non-responsibility to Aurora B. For the further structural modifications on the lateral chain of compound 19d, compounds 24 with longer lateral chain were designed and synthesized for testing leukemia cell lines. However, compounds 24 was significantly decrease inhibition potency against leukemia cell lines. Based on the in-vitro results, compounds 17b and 19d could be considered to be the best potential lead drug in our study for the development of new and effective therapies for leukemia treatment. On the other hand, the DHFR inhibition results indicated compound 19d possessed good inhibitory activity and better than the reported naphthalene derivative. Through further comparisons of the model superposition of three-dimensional (3D) conformations in DHFR, compound 19d presented a similar structural alignment to Methotrexate and the reported naphthalene derivative and led to similar drug-like functional relationships. As a results, compound 19d would be a potential DHFR inhibitor for anti-leukemia drug candidate.

HLA peptide-binding pocket diversity modulates immunological complications after cord blood transplant in acute leukaemia.

Pocket motifs and their amino acid positions of HLA molecules are known to govern antigen presentation to effector cells. Our objective was to analyse their influence on the risk of graft-versus-host disease (GVHD) and relapse after umbilical cord blood transplant (UCBT). The transplant characteristics of 849 patients with acute leukaemia were obtained from the Eurocord/EBMT database. Higher acute (a) GVHD was associated with homozygosity of UCB HLA-C amino acid positions 77 and 80 (NN/KK) (p = 0.008). Severe aGVHD was associated with HLA-A pocket B YSAVMENVHY motif (p = 0.002) and NN and RR genotypes of the HLA-C amino acid positions 77 and 156 (p = 0.006 and p = 0.002). Such risk was also increased in case of recipient and UCB mismatches in P4 (p < 0.0001) and P9 (p = 0.003) pockets of HLA-DQB1 alleles. For chronic GVHD, the pocket B YYAVMEISNY motif of the HLA-B*15:01 allele and the absence of mismatch between recipient and UCB in the P6 pocket of HLA-DRB1 were associated with a lower risk (p = 0.0007 and p = 0.0004). In relapse, both UCB pocket B YFAVMENVHY belonging to HLA-A*32:01 and recipient pocket B YDSVGENYQY motif of the HLA-C*07:01 allele were associated with higher risk (p = 0.0026 and p = 0.015). We provide clues on HLA-mediated cellular interactions and their role in the development of GVHD and relapse.

Sonrotoclax overcomes BCL2 G101V mutation–induced venetoclax resistance in preclinical models of hematologic malignancy

AbstractVenetoclax, the first-generation inhibitor of the apoptosis regulator B-cell lymphoma 2 (BCL2), disrupts the interaction between BCL2 and proapoptotic proteins, promoting the apoptosis in malignant cells. Venetoclax is the mainstay of therapy for relapsed chronic lymphocytic leukemia and is under investigation in multiple clinical trials for the treatment of various cancers. Although venetoclax treatment can result in high rates of durable remission, relapse has been widely observed, indicating the emergence of drug resistance. The G101V mutation in BCL2 is frequently observed in patients who relapsed treated with venetoclax and sufficient to confer resistance to venetoclax by interfering with compound binding. Therefore, the development of next-generation BCL2 inhibitors to overcome drug resistance is urgently needed. In this study, we discovered that sonrotoclax, a potent and selective BCL2 inhibitor, demonstrates stronger cytotoxic activity in various hematologic cancer cells and more profound tumor growth inhibition in multiple hematologic tumor models than venetoclax. Notably, sonrotoclax effectively inhibits venetoclax-resistant BCL2 variants, such as G101V. The crystal structures of wild-type BCL2/BCL2 G101V in complex with sonrotoclax revealed that sonrotoclax adopts a novel binding mode within the P2 pocket of BCL2 and could explain why sonrotoclax maintains stronger potency than venetoclax against the G101V mutant. In summary, sonrotoclax emerges as a potential second-generation BCL2 inhibitor for the treatment of hematologic malignancies with the potential to overcome BCL2 mutation–induced venetoclax resistance. Sonrotoclax is currently under investigation in multiple clinical trials.

A Novel Selective BCL2 Inhibitor with Limited Immune Suppression and Improved Safety Compared to Venetoclax

BCL2 is a key pro-survival protein that is overexpressed in many cancers. The first generation BCL2 inhibitor venetoclax has proved highly effective in multiple hematological malignancies including CLL and AML. However, the pharmacokinetic half-life of venetoclax is 26 hours which prevents optimization of the therapeutic window. Additionally, venetoclax metabolism is influenced by Cyp3A/4 inhibitors making their use in diseases where anti-fungal prophylaxis is required challenging. Venetoclax treatment can also result in significant immunosuppression including dose-limiting neutropenia and thrombocytopenia, and the loss of multiple lymphocyte populations, due in part to its effect on BCL-xL. Herein we present the pre-clinical evaluation of a novel highly selective BCL2 inhibitor, ZE50-0134, with equivalent anti-tumor efficacy, improved safety profile, and limited impact on non-malignant immune populations. ZE50-0134 binds the P2 pocket of BCL2, thereby increasing selectivity toward BCL-2, ZE50-0134 showed a 4600-fold greater selectivity for BCL2 over BCL-xL compared to venetoclax with only an 84-fold greater selectivity. In vivo murine and canine pharmacokinetics showed that ZE50-0134 has a substantially shorter half-life than venetoclax suggesting feasibility for periodic pulse dosing to limit the on-target adverse effects of BCL2 inhibition. The ADME properties of ZE50-0134 were exceptional. An in vivo murine study examining the pharmacology of ZE50-0134 or venetoclax given with ketoconazole, a strong CYP3A inhibitor, was performed demonstrating a 0.84-fold decrease in AUC with ZE50-0134 vs. a 1.52-fold increase in AUC with venetoclax suggesting less Cyp3A influence on ZE50-0134. Toxicology in rats and dogs suggest a 19-fold margin between therapeutically effective dose and toxicity with ZE50-0134. Given this data, we sought to validate that ZE50-0134 has less impact on normal immune populations. Wild-type C57BL6/J mice were treated daily with 100 mg/kg ZE50-0134, 100 mg/kg venetoclax or vehicle. A comprehensive 36-color spectral flow cytometry immune profiling panel was used to evaluate the effect of BCL2 inhibitors on multiple immune populations including B, T and NK cells as well as monocyte/macrophage and dendritic cell subsets in the peripheral blood. Venetoclax treated mice demonstrated a significant loss of multiple lymphocyte populations, including B, T, and NK cells, in the peripheral blood. Particularly, from Day 14 venetoclax treatment significantly reduced the percentage and absolute count of CD8 T cells (Figure 1). Some reduction in CD8 T cells was also observed with ZE50-0134 treatment, however it was significantly less than with venetoclax. No changes in the frequency of any normal myeloid population were observed. We next sought to examine the effectiveness of ZE50-0134 in B-cell and myeloid malignancies. Primary CLL cells were treated for 4 and 24 hours, corresponding to the expected half-life of ZE50-0134 and venetoclax, followed by washout. Cytotoxicity at 72 hours for both ZE50-0134 and venetoclax were equivalent for both short and prolonged treatment. We then assessed ZE50-0134 and venetoclax in vivo at 100 mg/kg in the RSV4;11 subcutaneous lymphoid model where we observed similar tumor growth regression. In AML, venetoclax is typically not effective as monotherapy and if often used in combination with azacytidine. To assess the relative efficacy of venetoclax and ZE50-0134 in combination with Azacitidine (5-Aza), we used the MOLM-13 cell line disseminated xenograft model. NCG mice were engrafted with MOLM-13 cells via the tail vein. From day 3, mice were treated with daily oral gavage of venetoclax or ZE50-0134 (100 mg/kg) alone or in combination with weekly intravenous 5-Aza (2.5 mg/kg). ZE50-0134 and venetoclax performed equally as single agents (median survival 23.5 days vs 22.5 days) and in combination with 5-Aza (median survival 35 days vs 35 days) (Figure 2). Together, this data suggests that ZE50-0134 is a highly effective BCL2 inhibitor with substantially less impact on the normal immune cells responsible for both anti-tumor and anti-infectious immunity. The pharmacology of this agent makes it an ideal therapeutic to optimize tumor BCL2 dependence and avoid untoward toxicity that occurs through continuous BCL2 targeting. This data supports the further clinical development of ZE50-0134 in both B cell malignancies and AML.

Small-Molecule Inhibition of KRAS through Conformational Selection.

Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] binds to KRAS with low micro-molar affinity [Chem. Biol. Drug Des.2019; 94(2):1441-1456]. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.

Influence of amino acid size at the P3 position of N-Cbz-tripeptide Michael acceptors targeting falcipain-2 and rhodesain for the treatment of malaria and human african trypanosomiasis

In recent decades, several structure–activity relationship (SAR) studies provided potent inhibitors of the cysteine proteases falcipain-2 (FP-2) and rhodesain (RD) from Plasmodium falciparum and Trypanosoma brucei rhodesiense, respectively. Whilst the roles of the warhead and residues targeting the P1 and P2 pockets of the proteases were extensively investigated, the roles of the amino acids occupying the S3 pocket were not widely assessed. Herein we report the synthesis and biological evaluation of a set of novel Michael acceptors bearing amino acids of increasing size at the P3 site (1a-g/2a-g, SPR20-SPR33) against FP-2, RD, P. falciparum, and T. brucei. Overall, the Michael acceptors bearing small amino acids at the P3 site exhibited the most potent inhibitory properties towards FP-2. In contrast, analogues with bulky residues at the P3 position were very potent rhodesain inhibitors. In cell based assays, single-digit micromolar EC50 values against the two protozoa were observed. These findings can be a starting point for the development of peptide-based FP-2 and RD inhibitors.

Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.

Discovery of N-sulfonylated aminosalicylic acids as dual MCL-1/BCL-xL inhibitors.

The anti-apoptotic protein MCL-1, which is overexpressed in multiple cancers, is presently a focus for the development of targeted drugs in oncology. We previously discovered inhibitors of MCL-1 based on 1-sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acids ("1,6-THQs"). However, with the nitrogen atom constrained in the bicyclic ring, we were unable to modify the alkyl portion of the tertiary sulfonamide functionality. Moreover, the introduction of additional functional groups onto the benzene ring portion of the THQ bicycle would not be trivial. Therefore, we elected to deconstruct the piperidine-type ring of the 6-carboxy-THQ lead to create a new 4-aminobenzoic acid scaffold. Given its simplicity, this permitted us to introduce diversity at the sulfonamide nitrogen, as well as vary the positions and substituents of the benzene ring. One of our most potent MCL-1 inhibitors, 6e-OH, exhibited a K i of 0.778 μM. Heteronuclear single quantum coherence experiments suggested 6e-OH bound in the canonical BH3-binding groove, with significant perturbations of R263, which forms a salt bridge with MCL-1's pro-apoptotic binding partners, as well as residues in the p2 pocket. Selectivity studies indicated that our compounds are dual inhibitors of MCL-1 and BCL-xL, with 17cd the most potent dual inhibitor: K i = 0.629 μM (MCL-1), 1.67 μM (BCL-xL). Whilst selective inhibitors may be more desirable in certain instances, polypharmacological agents whose additional target(s) address other pathways associated with the disease state, or serve to counter resistance mechanisms to the primary target, may prove particularly effective therapeutics. Since selective MCL-1 inhibition may be thwarted by overexpression of sister anti-apoptotic proteins, including BCL-xL and BCL-2, we believe our work lays a solid foundation towards the development of multi-targeting anti-cancer drugs.

1-Sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acids as simple, readily-accessible MCL-1 inhibitors.

MCL-1 is a member of the BCL-2 family of proteins that regulates the mitochondrial pathway of apoptosis. Overexpression of MCL-1 is associated with the development and progression of a range of human cancers, and is also responsible for the onset of resistance to conventional chemotherapies. Although several MCL-1 inhibitors have now advanced to clinical trials, recent suspensions and terminations reveal the urgency with which new inhibitor chemotypes must be discovered. Building on our previous studies of a chiral, isomeric lead, we report the discovery of a new chemotype to inhibit MCL-1: 1-sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acid. The nature of the sulfonyl moiety contributed significantly to the resulting inhibitory ability. For example, transforming a phenylsulfonyl group into a 4-chloro-3,5-dimethylphenoxy)phenyl)sulfonyl moiety elicited more than a 73-fold enhancement in inhibiton of MCL-1, possibly through targeting the p2 pocket in the BH3-binding groove, and so it is anticipated that further structure-activity studies here will lead to continued improvements in binding. It should be underscored that this class of MCL-1 inhibitors is readily accessible in four simple steps, is achiral and offers many avenues for optimization, all factors that are welcomed in the search for safe and effective inhibitors of this driver of cancer cell survival.

A computational perspective for tailor-made selective Mcl-1 and Bcl-XL inhibitors

In view of the crucial role of Mcl-1 in tumor survival and resistance as well as the importance of Bcl-XL in platelets homeostasis, an appropriate tuning of binding selectivity between Mcl-1 and Bcl-XL is crucial to discover safer anticancer candidates. In this respect, comprehensive computational approaches such as protein comparison, molecular docking, molecular dynamics simulations, mutagenesis, and density functional theory (DFT) calculation were applied to elucidate the different binding modes between highly selective Mcl-1 and Bcl-XL inhibitors. It was found that although Mcl-1 and Bcl-XL shared high sequence homology in their active pockets, the P2 pocket of Mcl-1 merged with P1 and P3, whereas the P3 pocket bridged through the P2 and P4 pocket of Bcl-XL. Accordingly, the key residues that determined the selectivity between Mcl-1 and Bcl-XL included Asn260, Arg263, and Phe270 in Mcl-1, and Glu96, Ser106, Leu108, Asn136, and Arg139 in Bcl-XL. In particular, Phe270 served as a major contributor to the π-π stacking interactions between Mcl-1 and its selective inhibitor, whereas Tyr195 was the key residue establishing the π−π stacking between Bcl-XL and its selective inhibitor. Collectively, these data shed promising light in depicting the selective mechanisms between Mcl-1 and Bcl-XL, which would lay a solid foundation for the future designing of the selective inhibitors in the discovery and optimization of a compound on the path toward developing future anticancer drugs.

Defining substrate selection by rhinoviral 2A proteinase through its crystal structure with the inhibitor zVAM.fmk

Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.

Abstract 1285: In vitro characterization of sotorasib and other RAS ‘His95-groove' binders and investigation of resistance mechanisms

Abstract Sotorasib (formerly known as AMG 510), the first-in-class KRASG12C inhibitor, has demonstrated promising clinical efficacy in KRAS p.G12C mutant cancers. Sotorasib binds to KRASG12C through a unique interaction with a surface groove created by side-chain rotation of histidine 95 (His95). Characterization of sotorasib and other His95-groove binders revealed enhanced potency and selectivity as compared to other KRASG12C inhibitor scaffolds, which bind in the P2 pocket via hydrogen bonding with His95. The novel binding mode of sotorasib also translated to similar biochemical and cellular potencies against both NRASG12C and HRASG12C, which encode leucine and glutamine at position 95, respectively. In contrast, other KRASG12C inhibitor scaffolds demonstrated a dramatic loss of potency against NRASG12C and HRASG12C, suggesting that the alternate residues impacted the binding of these molecules in the P2 pocket. To extend characterization of the cellular effects of RAS ‘His95-groove' binders, we analyzed the expression of major histocompatibility complex (MHC) class I proteins and other inflammatory markers in multiple human and murine KRAS p.G12C cell lines. These studies revealed a partial dependency on the cytosolic DNA-sensing (cGAS/STING) pathway for the effects observed with some markers. Finally, His95-groove binders were evaluated for potential mechanisms of resistance to this class of KRASG12C inhibitors. In the mouse syngeneic Lewis Lung Carcinoma (LL/2) cell line, which carries both KRAS p.G12C and NRAS p.Q61H mutations, intrinsic resistance to KRASG12C inhibition was observed, but combination treatment with the MEK inhibitor trametinib demonstrated synergistic improvement in the effects on viability. MIA PaCa-2 and NCI-H358 models of acquired resistance to KRASG12C inhibition were also developed through long-term exposure to high concentrations of sotorasib. Characterization of these resistant cell lines indicated a requirement for constant exposure to sotorasib and also showed that the resistance was not due to genetic alterations but involved either overexpression of KRAS or bypass signaling through alternative pathways. Taken together, these data demonstrate that His95-binders like sotorasib display superior potency and off-target selectivity, as well as unique activity against all versions of RASG12C. In addition, characterization of potential resistance mechanisms to sotorasib will inform combination strategies in the clinic. Citation Format: Anne Y. Saiki, Deanna Mohn, Yu Li, Tao Osgood, Karen Rex, Hui-Ling Wang, Ivonne Archibeque, Christopher Mohr, Pragathi Achanta, Andres Plata Stapper, Aaron S. Rapaport, Jude Canon, Victor J. Cee, Brian A. Lanman, J. Russell Lipford. In vitro characterization of sotorasib and other RAS ‘His95-groove' binders and investigation of resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1285.

Structural Comparison Between MHC Classes I and II; in Evolution, a Class-II-Like Molecule Probably Came First.

Structures of peptide-loaded major histocompatibility complex class I (pMHC-I) and class II (pMHC-II) complexes are similar. However, whereas pMHC-II complexes include similar-sized IIα and IIβ chains, pMHC-I complexes include a heavy chain (HC) and a single domain molecule β2-microglobulin (β2-m). Recently, we elucidated several pMHC-I and pMHC-II structures of primitive vertebrate species. In the present study, a comprehensive comparison of pMHC-I and pMHC-II structures helps to understand pMHC structural evolution and supports the earlier proposed—though debated—direction of MHC evolution from class II-type to class I. Extant pMHC-II structures share major functional characteristics with a deduced MHC-II-type homodimer ancestor. Evolutionary establishment of pMHC-I presumably involved important new functions such as (i) increased peptide selectivity by binding the peptides in a closed groove (ii), structural amplification of peptide ligand sequence differences by binding in a non-relaxed fashion, and (iii) increased peptide selectivity by syngeneic heterotrimer complex formation between peptide, HC, and β2-m. These new functions were associated with structures that since their establishment in early pMHC-I have been very well conserved, including a shifted and reorganized P1 pocket (aka A pocket), and insertion of a β2-m hydrophobic knob into the peptide binding domain β-sheet floor. A comparison between divergent species indicates better sequence conservation of peptide binding domains among MHC-I than among MHC-II, agreeing with more demanding interactions within pMHC-I complexes. In lungfishes, genes encoding fusions of all MHC-IIα and MHC-IIβ extracellular domains were identified, and although these lungfish genes presumably derived from classical MHC-II, they provide an alternative mechanistic hypothesis for how evolution from class II-type to class I may have occurred.

Structure-Based Design of A-1293102, a Potent and Selective BCL-XL Inhibitor.

BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

Structure-Guided Development of Potent Benzoylurea Inhibitors of BCL-XL and BCL-2.

The BCL-2 family of proteins (including the prosurvival proteins BCL-2, BCL-XL, and MCL-1) is an important target for the development of novel anticancer therapeutics. Despite the challenges of targeting protein-protein interaction (PPI) interfaces with small molecules, a number of inhibitors (called BH3 mimetics) have entered the clinic and the BCL-2 inhibitor, ABT-199/venetoclax, is already proving transformative. For BCL-XL, new validated chemical series are desirable. Here, we outline the crystallography-guided development of a structurally distinct series of BCL-XL/BCL-2 inhibitors based on a benzoylurea scaffold, originally proposed as α-helix mimetics. We describe structure-guided exploration of a cryptic "p5" pocket identified in BCL-XL. This work yields novel inhibitors with submicromolar binding, with marked selectivity toward BCL-XL. Extension into the hydrophobic p2 pocket yielded the most potent inhibitor in the series, binding strongly to BCL-XL and BCL-2 (nanomolar-range half-maximal inhibitory concentration (IC50)) and displaying mechanism-based killing in cells engineered to depend on BCL-XL for survival.

HLA‐DRB1 molecules and the presentation of anchor peptides from RhD, RhCE, and KEL proteins

Antigens from the Rh and Kell systems are recognized as the most immunogenic in clinical practice. This study evaluated the possible molecular mechanisms involved in the interaction of antigenic peptides with the DRB1 molecules, which help to explain the high frequency of anti-K and association of D + C antibodies in transfusion and incompatible pregnancy. We included 201 patients with antibodies against antigens from the Rh and Kell systems and compare them with 174,015 controls. HLA-DRB1 genotyping and in silico analysis were performed. The NetMHCIIpan software was used to identify RhD-, RhCE-, and KEL-derived anchor peptides that bind to DRB1 molecules. HLA-DRB1*15 is associated with an increased risk of D, C, E, and K alloimmunization, while the HLA-DRB1*01 and *12 alleles are overrepresented in patients with anti-C and anti-D, respectively. In silico analysis showed that three polymorphic points (60I, 68S, and 103S) common to C and D antigens can be presented by several DRB1 molecules, including DRB1*15:01. The DRB1*09:01 molecule, although not showing statistical significance, was able to interact strongly with almost all five anchor peptides from the sequence containing the polymorphic determinants of E antigen, except 217-WMFWPSVNS-225. The DRB1*15 molecule has specific physicochemical characteristics in residues 11P and 13R in the P4 pocket that can favor the response to various antigenic peptides. Anti-K alloimmunization is unrestricted for interaction with specific DRB1 molecules, which suggests that almost all individuals in our population have DRB1 molecules capable of binding to KEL-derived anchor peptides and produce anti-K when stimulated.

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-μM concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 μM: manidipine (4.8 μM), boceprevir (5.4 μM), lercanidipine (16.2 μM), bedaquiline (18.7 μM), and efonidipine (38.5 μM). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1′, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.