Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.

Pharmacological inhibition of noncanonical EED-EZH2 signaling overcomes chemoresistance in prostate cancer.

Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance. Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models. Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models. Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.

STAT3-activated lncRNA XIST accelerates the inflammatory response and apoptosis of LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe lung respiratory failure characterized by high morbidity and mortality. Novel findings demonstrated the critical roles of long non-coding RNA (lncRNA) in ALI. Here, we tried to investigate the roles and potential mechanism of lncRNA X-inactive specific transcript (XIST) in ALI. Results illustrated that lncRNA XIST was up-regulated in the lipopolysaccharide (LPS)-induced ALI mice models and pulmonary endothelial cells. Biofunctional assays unveiled that knockdown of XIST repressed the inflammatory response and apoptosis in LPS-induced endothelial cells. Mechanistically, XIST acted as the miR-146a-5p sponge to positively regulate STAT3. Moreover, STAT3 combined the promoter region of XIST to accelerate the transcription, constituting the positive feedback loop of XIST/miR-146a-5p/STAT3 in ALI. Collectively, these findings suggested that XIST knockdown attenuates the LPS-induced ALI, providing a potential therapeutic target.

Atractylenolide I enhances responsiveness to immune checkpoint blockade therapy by activating tumor antigen presentation.

One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.

Design and Synthesis of Fragment Derivatives with a Unique Inhibition Mechanism of the uPAR·uPA Interaction.

There is substantial interest in the development of small molecules that inhibit the tight and highly challenging protein-protein interaction between the glycophosphatidylinositol (GPI)-anchored cell surface receptor uPAR and the serine protease uPA. While preparing derivatives of a fragment-like compound that previously emerged from a computational screen, we identified compound 5 (IPR-3242), which inhibited binding of uPA to uPAR with submicromolar IC50s. The high inhibition potency prompted us to carry out studies to rule out potential aggregation, lack of stability, reactivity, and nonspecific inhibition. We designed and prepared 16 derivatives to further explore the role of each substituent. Interestingly, the compounds only partially inhibited binding of a fluorescently labeled α-helical peptide that binds to uPAR at the uPAR·uPA interface. Collectively, the results suggest that the compounds bind to uPAR outside of the uPAR·uPA interface, trapping the receptor into a conformation that is not able to bind to uPA. Additional studies will have to be carried out to determine whether this unique inhibition mechanism can occur at the cell surface.

m6 A transferase METTL3-induced lncRNA ABHD11-AS1 promotes the Warburg effect of non-small-cell lung cancer.

N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the pathological roles of m6 A and lncRNAs in NSCLC progression are still limited and undefined. Here, lncRNA ABHD11-AS1 was upregulated in NSCLC tissue specimens and cells and the ectopic overexpression was closely correlated with unfavorable prognosis of NSCLC patients. Functionally, ABHD11-AS1 promoted the proliferation and Warburg effect of NSCLC. Mechanistically, m6 A profile was analyzed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). MeRIP-Seq presented that there was m6 A modification site in ABHD11-AS1. m6 A methyltransferase-like 3 (METTL3) installed the m6 A modification and enhanced ABHD11-AS1 transcript stability to increase its expression. In conclusion, our findings highlight the function and mechanism of METTL3-induced ABHD11-AS1 in NSCLC and inspire the understanding of m6 A and lncRNA in cancer biology.

Synthesis and Hydration Characteristic of Geopolymer Based on Lead Smelting Slag.

Lead smelting slag (LSS) has been identified as general industrial solid waste, which is produced from the pyrometallurgical treatment of the Shuikoushan process for primary lead production in China. The LSS-based geopolymer was synthesized after high-energy ball milling. The effect of unconfined compressive strength (UCS) on the synthesis parameters of the geopolymer was optimized. Under the best parameters of the geopolymer (modulus of water glass was 1-1.5, dosage of water glass (W(SiO2+Na2O)) was 5% and water-to-binder ratio was 0.2), the UCS reached 76.09 MPa after curing for 28 days. The toxicity characteristic leaching procedure (TCLP) leaching concentration of Zn from LSS fell from 167.16 to 93.99 mg/L after alkali-activation, which was below the limit allowed. Meanwhile, C-S-H and the geopolymer of the hydration products were identified from the geopolymer. In addition, the behavior of iron was also discussed. Then, the hydration process characteristics of the LSS-based geopolymer were proposed. The obtained results showed that Ca2+ and Fe2+ occupied the site of the network as modifiers in the glass phase and then dissociated from the glass network after the water glass activation. At the same time, C-S-H, the geopolymer and Fe(OH)2 gel were produced, and then the Fe(OH)2 was easily oxidized to Fe(OH)3 under the air curing conditions. Consequently, the conclusion was drawn that LSS was an implementable raw material for geopolymer production.

Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases.

Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.

Effect of simulated acid rain on stability of arsenic calcium residue in residue field.

In recent years, acid rain had a serious negative impact on the leaching behavior of industrial waste residue. Researches were mainly focused on the environmental hazards of heavy metal in the leachate, but ignored the effects of heavy metal speciation on the stability of waste residue in the subsequent stabilization process. In this study, the unstable calcium-arsenic compounds in the arsenic calcium residue were firstly removed by leaching process; subsequently, the crystallization agent was added to treat the remaining calcium-arsenic mixture. The results of the leaching process demonstrated that the decrease in particle size and pH value directly affected the increase in the cumulative leaching amount of arsenic, and the cumulative leaching ratio reached 1.55%. In addition, the concentration of arsenic decreased from 3583 to 49.1 mg L-1. After the crystallization process, the arsenic concentration was lower than the limit value of Identification Standards for Hazardous Wastes (GB 5085.3-2007). The SEM analysis showed the bulk structures, and XRD pattern confirmed that they were the stable compounds. Moreover, the result of XRD and SEM illustrated that acid concentration, chloride ions and sulfate ions were contributed to the transformation and growth of stable calcium arsenate compounds. Therefore, effective control of the acidity of acid rain, the type of anions in acid rain, and the particle size of residues would contribute to adjusting the arsenic speciation to be more stable. The leaching-crystallization process was of great significance to improve the stability of the arsenic-containing residue.

Cotreatment of MSWI Fly Ash and Granulated Lead Smelting Slag Using a Geopolymer System.

Municipal solid waste incineration fly ash (MSWI FA) and granulated lead smelting slag (GLSS) are toxic industrial wastes. In the present study, granulated lead smelting slag (GLSS) was pretreated as a geopolymer precursor through the high-energy ball milling activation process, which could be used as a geopolymeric solidification/stabilization (S/S) reagent for MSWI FA. The S/S process has been estimated through the physical properties and heavy metals leachability of the S/S matrices. The results show that the compressive strength of the geopolymer matrix reaches 15.32 MPa after curing for 28 days under the best parameters, and the physical properties meet the requirement of MU10 grade fly ash brick. In addition, the toxicity characteristic leaching procedure (TCLP) test results show that arsenic and heavy metals are immobilized effectively in the geopolymer matrix, and their concentrations in the leachate are far below the US EPA TCLP limits. The hydration products of the geopolymer binder are characterized by X-ray diffraction and Fourier transform infrared methods. The results show that the geopolymer gel and Friedel's salt are the main hydration products. The S/S mechanism of the arsenic and heavy metals in the geopolymer matrix mainly involves physical encapsulation of the geopolymer gel, geopolymer adsorption and ion exchange of Friedel's salt.

Formation mechanism of zinc-doped fayalite (Fe2-xZnxSiO4) slag during copper smelting.

The interactions between Fe2SiO4 and ZnO play an essential role in the recovery of zinc from copper slag. The dissolution and substitution mechanism of ZnO in fayalite were investigated by using TG-DSC, XRD, PPMS DynaCool, XPS, Mossbauer and SEM-EDS analyses and compared with MS calculation results. The results indicate that the dissolution and substitution are actually processes of the penetrating dissolution of Zn(II) ions that can be divided into three steps: 1) ZnO dissociates into Zn1-yO and Zn(II) ions; 2) Zn(II) penetrates the gap of the octahedron outer layer to substitute Fe(II) sites in the internal structure of SiOFe(II) (M2) to form (Fe2-x, Znx)SiO4; 3) Fe(II) is forced to migrate to the surface of (Fe2-x, Znx)SiO4 to form (Zn1-y, Fe(II)y)O. These findings can be derived the occurrence state and distribution of zinc in copper slag theoretically.

Preparation of red mud-based geopolymer materials from MSWI fly ash and red mud by mechanical activation.

A novel method to activate red mud was proposed in this study. Municipal solid waste incineration fly ash (MSWIFA) and red mud were utilized to prepare red mud-based geopolymer materials (RGM). The hydration characteristics of RGM were studied by X-ray diffraction, scanning electronic microscopy, and Fourier transform infrared spectroscopy. The long-term stability and physical properties of RGM were tested by freeze-thaw cycle, European Community Bureau reference (BCR) and unconfined compressive strength (UCS) tests. Results showed that mechanical activation can not only effectively activate red mud, but also effectively improve the reaction of MSWIFA and red mud. When 14% sodium silicate was added to the binder, the UCS reached 12.75 MPa at 28 days. In the RGM, aluminosilicate was effectively activated by mechanical activation and reacted with calcium ion to form complex hydration products. The activator reacts adequately with activated aluminum to form a high-strength geopolymer. The freeze-thaw cycles and BCR test results also showed that the RGM had long-term stability and the characteristics satisfied the requirements of MU10 fly ash bricks. This study demonstrated that RGM may be utilized in cement composites.

Small molecules inhibit ex vivo tumor growth in bone.

Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.

Small-molecule CaVα1⋠CaVß antagonist suppresses neuronal voltage-gated calcium-channel trafficking.

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and ß subunits. The high-affinity CaV2.2α1⋅CaVß3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVß3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVß3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.

Epithelial-mesenchymal transition and GALC expression of circulating tumor cells indicate metastasis and poor prognosis in non-small cell lung cancer.

BACKGROUND: Circulating tumor cells (CTCs) is a promising biomarker for cancer prognosis and monitoring. Molecular characterizing of CTCs could provide beneficial information on the basis of CTCs counting. OBJECTIVE: To investigate the epithelial-mesenchymal transition (EMT) phenotypes and GALC mRNA expression of CTCs in non-small cell lung cancer (NSCLC) patients. METHODS: We analyzed the baseline number, EMT classification, and GALC expression of CTCs in 47 NSCLC patients using CanPatrol platform and RNA in situ hybridization technique. RESULTS: CTCs were detected in 91.5% patients ranging 0-47/5 mL blood. Increased CTCs were associated with advanced tumor stages (6/5 mL) compared with early stages (3.5/5 mL). Patients with effective treatment response presented lower CTCs (3.5/5 mL) than patients with insufficient response (7/5 mL). Epithelial, hybrid and mesenchymal CTCs were detected in 55.4%, 78.7% and 61.7% patients, respectively. Patients with distant metastasis and poor curative outcomes presented higher level of EMT-CTCs. GALC expression was positive in CTCs of 80.6% patients and closely correlated with tumor number and distant metastasis and treatment outcomes. CONCLUSIONS: EMT phenotypes and GALC expression of CTCs are correlated with cancer metastasis and therapeutic outcomes, suggesting them to be potential markers for the prognosis of NSCLC.

The effect of foxp3-overexpressing Treg cells on non-small cell lung cancer cells.

The aim of the present study was to investigate the novel mechanisms of forkhead box protein P3 (foxp3) in T regulatory (Treg) cells in lung cancer behavior. Treg cells were isolated from the peripheral blood of healthy volunteers and then co­cultured with 95D cells. A plasmid overexpressing foxp3 was constructed and transfected into Treg cells and an MTS assay was performed to assess cell viability. Flow cytometry was performed to evaluate cell apoptosis and reverse transcription­quantitative polymerase chain reaction was used to measure mRNA expression. A Transwell assay was used to assess cell invasion. Treg cells were successfully isolated from peripheral blood with purity of 94.26%. Foxp3 expression in Treg cells was significantly increased following co­culture with 95D cells, while matrix metalloproteinase­9 expression was upregulated in 95D cells co­cultured with Treg cells. The apoptosis, invasion and migration abilities of 95D cells were suppressed by co­culture with Treg cells, whereas the adhesive ability was enhanced. Foxp3 overexpression in Treg cells enhanced the viability and invasiveness of 95D cells, whereas cell adhesion and migration were decreased. The results of the present study demonstrate that the viability and invasiveness of 95D cells are enhanced by foxp3 overexpression in Treg cells, indicating that increased levels of foxp3 in the tumor microenvironment may promote tumor cell growth.

Utilization of red mud and Pb/Zn smelter waste for the synthesis of a red mud-based cementitious material.

A new method in which Pb/Zn smelter waste containing arsenic and heavy metals (arsenic sludge), red mud and lime are utilized to prepare red mud-based cementitious material (RCM) is proposed in this study. XRD, SEM, FTIR and unconfined compressive strength (UCS) tests were employed to assess the physicochemical properties of RCM. In addition, ettringite and iron oxide-containing ettringite were used to study the hydration mechanism of RCM. The results show that the UCS of the RCM (red mud+arsenic sludge+lime) was higher than that of the binder (red mud+arsenic sludge). When the mass ratio of m (binder): m (lime) was 94:6 and then maintained 28days at ambient temperature, the UCS reached 12.05MPa. The red mud has potential cementitious characteristics, and the major source of those characteristics was the aluminium oxide. In the red mud-arsenic sludge-lime system, aluminium oxide was effectively activated by lime and gypsum to form complex hydration products. Some of the aluminium in ettringite was replaced by iron to form calcium sulfoferrite hydrate. The BCR and leaching toxicity results show that the leaching concentration was strongly dependent on the chemical speciation of arsenic and the hydration products. Therefore, the investigated red mud and arsenic sludge can be successfully utilized in cement composites to create a red mud-based cementitious material.

Co-treatment of flotation waste, neutralization sludge, and arsenic-containing gypsum sludge from copper smelting: solidification/stabilization of arsenic and heavy metals with minimal cement clinker.

Flotation waste of copper slag (FWCS), neutralization sludge (NS), and arsenic-containing gypsum sludge (GS), both of which are difficult to dispose of, are major solid wastes produced by the copper smelting. This study focused on the co-treatment of FWCS, NS, and GS for solidification/stabilization of arsenic and heavy metals with minimal cement clinker. Firstly, the preparation parameters of binder composed of FWCS, NS, and cement clinker were optimized to be FWCS dosage of 40%, NS dosage of 10%, cement clinker dosage of 50%, mill time of 1.5 h, and water-to-binder ratio of 0.25. On these conditions, the unconfined compressive strength (UCS) of the binder reached 43.24 MPa after hydration of 28 days. Then, the binder was used to solidify/stabilize the As-containing GS. When the mass ratio of binder-to-GS was 5:5, the UCS of matrix can reach 11.06 MPa after hydration of 28 days, meeting the required UCS level of MU10 brick in China. Moreover, arsenic and other heavy metals in FWCS, NS, and GS were effectively solidified or stabilized. The heavy metal concentrations in leachate were much lower than those in the limits of China standard leaching test (CSLT). Therefore, the matrices were potential to be used as bricks in some constructions. XRD analysis shows that the main hydration products of the matrix were portlandite and calcium silicate hydrate. These hydration products may play a significant role in the stabilization/solidification of arsenic and heavy metals.

Small molecules inhibit STAT3 activation, autophagy, and cancer cell anchorage-independent growth.

Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2, estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase (MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar IC50s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to evaluate its effect on STAT3 phosphorylation and DNA binding. The compound's inhibition of cell growth led to measurements of survivin, Bcl-XL, p21WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were quantified following treatment of cells with 6 to determine whether the compound affected autophagy, a process that is known to be activated by STAT3. Compound 6 provides a starting point for the development of small molecules with polypharmacology that can suppress TNBC growth and metastasis.

Indications of 5' to 3' Interbase Electron Transfer as the First Step of Pyrimidine Dimer Formation Probed by a Dinucleotide Analog.

Pyrimidine dimers are the most common DNA lesions generated under UV radiation. To reveal the molecular mechanisms behind their formation, it is of significance to reveal the roles of each pyrimidine residue. We thus replaced the 5'-pyrimidine residue with a photochemically inert xylene moiety (X). The electron-rich X can be readily oxidized but not reduced, defining the direction of interbase electron transfer (ET). Irradiation of the XpT dinucleotide under 254 nm UV light generates two major photoproducts: a pyrimidine (6-4) pyrimidone analog (6-4PP) and an analog of the so-called spore photoproduct (SP). Both products are formed by reaction at C4=O of the photo-excited 3'-thymidine (T), which indicates that excitation of a single "driver" residue is sufficient to trigger pyrimidine dimerization. Our quantum-chemical calculations demonstrated that photo-excited 3'-T accepts an electron from 5'-X. The resulting charge-separated radical pair lowers its energy upon formation of interbase covalent bonds, eventually yielding 6-4PP and SP.