Unveiling a Hidden Pocket in HIV-1 Protease: New Insights Into Retroviral Protease Cantilever-Tip Region Characteristics.

The HIV-1 protease is critical for the process of viral maturation and as such, it is one of the most well characterized proteins in the Protein Data Bank. There is some evidence to suggest that the HIV-1 protease is capable of accommodating small molecule fragments at several locations on its surface outside of the active site. However, some pockets on the surface of proteins remain unformed in the apo structure and are termed "cryptic sites." To date, no cryptic sites have been identified in the structure of HIV-1 protease. Here, we characterize a novel cryptic cantilever pocket on the surface of the HIV-1 protease through mixed-solvent molecular dynamics simulations using several probes. Interestingly, we noted that several homologous retroviral proteases exhibit evolutionarily conserved dynamics in the cantilever region and possess a conserved pocket in the cantilever region. Immobilization of the cantilever region of the HIV-1 protease via disulfide cross-linking resulted in curling-in of the flap tips and the propensity for the protease to adopt a semi-open flap conformation. Structure-based analysis and fragment-based screening of the cryptic cantilever pocket suggested that the pocket may be capable of accommodating ligand structures. Furthermore, molecular dynamics simulations of a top scoring fragment bound to the cryptic pocket illustrated altered flap dynamics of the fragment-bound enzyme. Together, these results suggest that the mobility of the cantilever region plays a key role in the global dynamics of retroviral proteases. Therefore, the cryptic cantilever pocket of the HIV-1 protease may represent an interesting target for future in vitro studies.

Nontarget Identification of Novel Organophosphorus Flame Retardants and Plasticizers in Indoor Air and Dust from Multiple Microenvironments in China.

The indoor environment is a typical source for organophosphorus flame retardants and plasticizers (OPFRs), yet the source characteristics of OPFRs in different microenvironments remain less clear. This study collected 109 indoor air samples and 34 paired indoor dust samples from 4 typical microenvironments within a university in Tianjin, China, including the dormitory, office, library, and information center. 29 target OPFRs were analyzed, and novel organophosphorus compounds (NOPs) were identified by fragment-based nontarget analysis. Target OPFRs exhibited the highest air and dust concentrations of 46.2-234 ng/m3 and 20.4-76.0 µg/g, respectively, in the information center, where chlorinated OPFRs were dominant. Triphenyl phosphate (TPHP) was the primary OPFR in office air, while tris(2-chloroethyl) phosphate dominated in the dust. TPHP was predominant in the library. Triethyl phosphate (TEP) was ubiquitous in the dormitory, and tris(2-butoxyethyl) phosphate was particularly high in the dust. 9 of 25 NOPs were identified for the first time, mainly from the information center and office, such as bis(chloropropyl) 2,3-dichloropropyl phosphate. Diphenyl phosphinic acid, two hydroxylated and methylated metabolites of tris(2,4-ditert-butylphenyl) phosphite (AO168), and a dimer phosphate were newly reported in the indoor environment. NOPs were widely associated with target OPFRs, and their human exposure risk and environmental behaviors warrant further study.

Nontarget Discovery of Per- and Polyfluoroalkyl Sulfonyl Halides in Soils by Integration of Derivatization and Specific Fragment-Based Liquid Chromatography-High Resolution Mass Spectrometry Screening.

Though long recognized as synthetic precursors to other poly- and perfluoroalkyl substances (PFASs), most poly- and perfluoroalkyl sulfonyl halides (PASXs) cannot be directly measured and have generally received minimal attention. Inspired by the redox reaction between sulfonyl halide groups and p-toluenethiol in organic chemistry, we developed a novel nontarget analysis strategy for PASXs by intergrating derivatization and specific fragment-based liquid chromatography-high resolution mass spectrometry screening for m/z 82.961 [SO2F-] and m/z 95.934 [S2O2-]. By using this strategy, we discovered 11 PASXs, namely, perfluoroalkyl sulfonyl fluorides (5), polyfluoroalkyl sulfonyl fluorides (2), unsaturated perfluoroalkyl sulfonyl fluoride (1), and perfluoroalkyl sulfonyl chlorides (3) in soil samples collected from an abandoned fluorochemical manufacturing park. These average ∑PASXs concentrations were 1120 µg kg-1 (range: 9.7-9860 µg kg-1), which were very likely to be the key intermediates and undesired byproducts of electrochemical fluorination processes. Spatial variation in the mass ratio of ∑PASXs to ∑PFSAs (range: 0.7-795%) also indicates their different transportation pathways. More importantly, the decline of PASXs and increase of perfluoroalkyl sulfonates (when compared to a prior study at this site) suggest the continued hydrolysis of PASXs and the relatively fast environmental transformation rates in the abandoned fluorochemical park soils. Overall, these findings demonstrated the utility of a novel nontarget analysis strategy, which may change most PASXs from inferred precursors to measured intermediates and further could be adapted for structures, distribution, and transformation studies of PFASXs in other matrices.

Ligandability assessment of the C-terminal Rel-homology domain of NFAT1.

Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.

An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

MALDI-TOF Mass Spectrometry-Based Assay for Measuring Covalent Target Engagement of KRAS G12C Inhibitors.

MALDI-TOF mass spectrometry enables high-throughput screening of covalent fragment libraries and SAR compound progressions of selective KRAS G12C inhibitors. Using the MALDI-TOF platform instead of the more traditional ESI-MS TOF/orbitrap instrumentation can radically shorten sample acquisition time, allowing up to 384 samples to be screened in 30 min. The typical throughput for a covalent library screen is 1152 samples per 8 h, including processing, calculation, and reporting steps. The throughput can be doubled without any significant assay modification.

Fragment-based discovery of small molecule inhibitors of the HDGFRP2 PWWP domain.

The PWWP domain of hepatoma-derived growth factor-related protein 2 (HDGFRP2) recognizes methylated histones to initiate the recruitment of homologous recombination repair proteins to damaged silent genes. The combined depletion of HDGFRP2 and its paralog PSIP1 effectively impedes the onset and progression of diffuse intrinsic pontine glioma (DIPG). Here, we discovered varenicline and 4-(4-bromo-1H-pyrazol-3-yl) pyridine (BPP) as inhibitors of the HDGFRP2 PWWP domain through a fragment-based screening method. The complex crystal structures reveal that both Varenicline and BPP engage with the aromatic cage of the HDGFRP2 PWWP domain, albeit via unique binding mechanisms. Notably, BPP represents the first single-digit micromolar inhibitor of the HDGFRP2 PWWP domain with a high ligand efficiency. As a dual inhibitor targeting both HDGFRP2 and PSIP1 PWWP domains, BPP offers an exceptional foundation for further optimization into a chemical tool to dissect the synergetic function of HDGFRP2 and PSIP1 in DIPG pathogenesis.

Optimizing NMR fragment-based drug screening for membrane protein targets.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.