Crystallographic Methods Research Articles

Multifunctional, fluorene-based modulator of cholinergic and GABAergic neurotransmission as a novel drug candidate for palliative treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and behavioral and psychological symptoms of dementia (BPSD). Given that cholinergic neurons are predominantly affected in AD, current treatments primarily aim to enhance cholinergic neurotransmission. However, imbalances in other neurotransmitters, such as γ-aminobutyric acid (GABA), also contribute to AD symptomatology. In the presented research, by using a combination of crystallography and computational methods we developed 6 as a dual modulator of GABAergic and cholinergic neurotransmission systems. Compound 6 demonstrated inhibition of BuChE (IC50 = 0.21 μM) and GABA transporter 1 (IC50 = 10.96 μM) and 3 (IC50 = 7.76 μM), along with a favorable drug-likeness profile. Subsequent in vivo studies revealed effectiveness of 6 in enhancing memory retention and alleviating anxiety and depression symptoms in animal models, while also proving safe and bioavailable for oral administration. The innovative multi-target-directed ligand 6 offers a new approach to treating cognitive deficits and BPSD in AD.

In vitro, in silico and crystallographic-based identification of serine protease inhibitors

Serine proteases are involved in various ailments, including pancreatitis, and colon cancer. Based on substrate recognition serine proteases are classified into different groups. Trypsin and trypsin-like serine proteases are among most studied group of serine proteases. Trypsin is among the chief hydrolysing enzyme involved in the pathogenesis of pancreatitis. Its inhibition can help to manage the disease. Herein, we investigated the trypsin inhibitory effect of some arginine-based small molecules, through in vitro, in silico, and crystallographic methods. Compounds 1-3 were evaluated against bovine pancreatic trypsin (BPT). Compound 1 was found to be active against trypsin with IC50 value of 247.98 ± 2.44 μM. Molecular docking studies were used to investigate the binding energy and binding conformation of inhibitor. All three compounds were subjected to crystallisation with trypsin. Compounds 1-2 were successfully crystallised with BPT The crystal structures of trypsin in complexed with compounds 1, and 2 were determined at 2.30 and 2.50 Å resolution, respectively. Both molecules showed their binding affinity with the active site residues of trypsin. This study will provide insight into the binding mechanism of E-64 and arginine and might be useful in designing effective inhibitors of serine proteases.

Ligand Identification in CryoEM and X-ray Maps Using Deep Learning.

Accurately identifying ligands plays a crucial role in the process of structure-guided drug design. Based on density maps from X-ray diffraction or cryogenic-sample electron microscopy (cryoEM), scientists verify whether small-molecule ligands bind to active sites of interest. However, the interpretation of density maps is challenging, and cognitive bias can sometimes mislead investigators into modeling fictitious compounds. Ligand identification can be aided by automatic methods, but existing approaches are available only for X-ray diffraction and are based on iterative fitting or feature-engineered machine learning rather than end-to-end deep learning. Here, we propose to identify ligands using a deep learning approach that treats density maps as 3D point clouds. We show that the proposed model is on par with existing machine learning methods for X-ray crystallography while also being applicable to cryoEM density maps. Our study demonstrates that electron density map fragments can aid the training of models that can later be applied to cryoEM structures but also highlights challenges associated with the standardization of electron microscopy maps and the quality assessment of cryoEM ligands.

Analysis of the Physical and Chemical Composition of Sludge from the Water Treatment Plant

The properties and composition of the sludge generated in water treatment systems depend primarily on the type and composition of the water to be treated, the treatment methods, and the type and doses of chemical reactants. The sludge produced in the water treatment plant (WTP) under study follows the technological processes of coagulation, flocculation, sedimentation, and filtration. The analyses aimed to characterize the sludge in terms of its physico-chemical properties and classify it in terms of its potential discharge into the river and management. Four series of sediment tests were conducted over a calendar year (March, June, September and December), analysing selected parameters using various test methods, including the X-ray crystallography (XRF) method. The publication's authors showed that the sediment consists mainly of sand, clay, and silt particles with grain sizes ranging from 0.001 mm to 1 mm. Silica (53.78%), alumina (23.58%), calcium oxide (8.28%), iron (III) oxide (5.61%), and potassium oxide (2.36%) represent the main chemical constituents present in the sediment. The authors characterized the sediment in terms of the content of biogenic compounds: various forms of phosphorus and nitrogen, organic compounds – determined as total organic carbon (TOC), selected metals, and the content of individual elements (carbon, hydrogen, oxygen, nitrogen, sulfur). In addition, the sludge samples were also characterized in terms of calorific value, ash content, water content, and heat of combustion. Discharging WTP sludge into rivers, ponds, and lakes or storing dewatered sludge is an environmentally unfriendly form of disposal for this type of waste. The authors see the possibility of conducting further research on using WTP sludge in wastewater treatment, removing heavy metals from aqueous solutions, producing cement and construction materials, and recovering or recycling.

Synthesis Techniques and Biomedical Applications of Cyclodextrin Metal-Organic Frameworks.

Cyclodextrin Metal-Organic Frameworks (CD MOFs) represent an innovative class of materials with remarkable properties and a broad range of applications. This review provides a comprehensive overview of the synthesis techniques, structural characterization, and diverse applications of CD-MOFs. By combining cyclodextrins (CDs) with metal-organic frameworks (MOFs), CD-MOFs are developed with enhanced functionality. The synthesis methods, including various metal sources, coordination modes, and post-synthesis modifications, are discussed alongside advanced structural characterization techniques like X-ray crystallography and spectroscopic methods. The unique characteristics of CD-MOFs, such as high specific surface area, tunable porosity, and customizable chemical structure, make them exceptional candidates for applications in gas adsorption, drug delivery, catalysis, sensing, and environmental remediation. Notably, CD-MOFs show significant promise as nanocarriers in drug delivery systems, offering improved therapeutic outcomes due to their efficient encapsulation and controlled release capabilities. The review highlights recent advancements and underscores the potential impact of CD-MOFs in driving future innovations across various scientific fields.

Supradecoration induced novel Properties: A comparative study of Ferrocene and Ferrocenyl α-aminophosphonate

Decorating chemical motifs with appropriate functional groups capable of supramolecular interactions hold key in modulating these novel systems for targeted applications. This work by means of a comparative study of Ferrocene and Ferrocenyl α- aminophosphonate highlight supradecoration induced multi paradigm properties across materials to medicine. The observed results indicate an increase in crystal framework energy with induction of mechanochromism and aggregation-induced emission in case of Ferrocenyl-α-amino phosphonate as novel supra decorated Ferrocene motif. Comparative BSA and BLC studies indicated improved biocompatibility with enhancement of catalase enzyme activity in case of Ferrocenyl-α- aminophosphonate compared to pristine Ferrocene. The relative quantification of intermolecular non covalent interactions of Ferrocene and Ferrocenyl-α- aminophosphonate evaluated using Quantum crystallographic methods and non-covalent interaction analysis were used to corroborate the supramodulation effects. The observed influence of Ferrocenyl α-aminophosphonate on the enzymatic activity of catalase was also examined using a comparative molecular docking analysis. The docking results corroborated influence of supradecoration on binding affinity and binding site, which are vital for designing ligands of enhanced catalase activity and also helpful in prediction of newer regulatory sites on enzyme. Taken together this work signifies how supra decoration of Ferrocene motif with functionalities capable of diverse intermolecular non-covalent interactions modulate molecular properties for newer applications.

Use of mixed saliva in the diagnosis of recurrent herpetic stomatitis in patients with dental problems: An interventional single-center, prospective, randomized, uncontrolled study

BACKGROUND: Recurrent herpetic stomatitis, also known as oral herpes, is one of the most common viral diseases of the oral cavity. Currently, saliva is widely used in innovative diagnostic methods. However, there are only limited data supporting the diagnostic value of salivary biomarkers in herpes infections. AIM: To establish the informative value of using the wedge-shaped dehydration method and the express method for determining salivary amylase activity in patients with recurrent herpetic stomatitis. MATERIALS AND METHODS: Diagnosis of recurrent herpes stomatitis was carried out based on the results of a clinical examination and confirmed using molecular genetic (PCR with real-time detection) and serological studies (ELISA — VectoHSV-IgG test system). For non-invasive diagnosis of recurrent herpetic stomatitis, we used crystallography methods — wedge-shaped dehydration of oral fluid and an express method for determining salivary amylase activity. The study included 26 patients with HHS in the acute stage (frequency of exacerbations of 4 or more per year) and 14 patients in the control group with no history of episodes of herpetic stomatitis. RESULTS: The results of crystallography of mixed saliva and determination of salivary amylase activity in patients diagnosed with recurrent herpes before and after treatment are presented. Specific changes in facies in the protein zone were established during wedge-shaped dehydration during the exacerbation period and an increase in salivary amylase activity in patients with recurrent herpetic stomatitis in the acute stage. CONCLUSION: To confirm the diagnosis of recurrent herpetic stomatitis at a dental appointment, a non-invasive method of wedge-shaped dehydration using samples of mixed saliva is recommended, and to determine the effectiveness of treatment, along with clinical indicators, the most informative is a rapid test based on a study of the level of amylase in mixed saliva.

Topological constraints-induced radiation shielding efficiency of SiO2 α-cristobalite polymorphism: Signatures from Hirshfeld pseudo-surfaces

The paper presents a synergetic and comprehensive investigation into γ-radiation shields properties on SiO2 α-cristobalite subject varying external pressures (0<p < 9.1 GPa). Utilizing crystallographic methods involving γ-rays and Hirshfeld pseudo-surface (HPSs) analyses, we explore the structural responses of SiO2 α-cristobalite to external pressure and its consequent impact on MAC and LAC. The study encompasses a broad energy range (0.015<E < 15.0 MeV), employing online tool for analysis. Our findings reveal that the fundamental shielding characteristics of SiO2 α-cristobalite, including (5.809<MAC<0.021 cm2/g), (3.10 × 1023<Neff< 3.74 × 1023 e/g), (10.32<Zeff<12.43), and (10.79<Zeq<11.56), remain reliable across different polymorphic forms within the energy domain and varying pressures. The LAC values exhibit a clear dependency on both incident photon energies where it's LAC ∈ [0.042 to 11.776] cm−1 as pressure elevated on the crystal. This relationship underscores the influence of pressure-induced changes in crystal density on LAC values. Additionally, changes in lattice parameters (4.975<a<4.599 Å) and bond lengths under pressure contribute to this observed variation in the LAC values. Further analysis of the topological constraints (TCs) (i.e., 1.603<Si⋯O < 1.610 Å, 111.42°<O⋯Si⋯O < 112.00°, and 146.49°<Si⋯O⋯Si < 127.80° bending and twisting angles respectively) reveals defined correlations with LAC values.HPS topological analyses were also employed shed light on valuable insights into the mutual forces of interaction between atoms in the crystal unit cell. Changes in external stress lead to transformations in the HPSs, reflecting the impact of pressure on topological constraints. The evolution of HPS volume (i.e., 179.59-35.24 Å3) and area (i.e., 205.73-161.51 Å2) as well as void's volume (i.e., 10.20-0.17 Å3) and surface area (i.e., 46.10-3.12 Å2) within the crystal lattice is also observed, indicating a direct relationship between HPSs, crystal compactness, electron density and radiation effectiveness. These signatures have been correlated with TCs and thus on the γ-rays shield effectiveness.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

Phyllotaxis: Leaf arrangement on a horizontal branch and crystallographic borders

Phyllotaxis is an orderly arrangement of leaves on plant stems and branches. The natural reason of this arrangement is that each species based on the habitat conditions in the course of evolution in one way or another solved the problem of optimising the light flux reaching each leaf and providing photosynthesis. The found optimum was fixed in the genotype and became a species phenotypic trait. In a scientific study, it should be recorded with a reliability that allows for small fluctuations inherent in plant forms under the influence of the environment. Crystallography methods are used to describe the arrangement of leaves. In the course of the study, it has been proposed to describe the arrangement of leaves on a horizontal branch and a vertical stem using the theory of crystallographic borders in the first case, and helical axes – in the second. When describing the arrangement of leaves on a horizontal branch, seven types of crystallographic borders turned out to be theoretically consistent. The nomenclature of types according to the generating operations of symmetry is considered unambiguously fixing the orthogonal increment of a symmetrical sheet (PT, ST, RPT, PT*) and oblique – symmetrical and asymmetrical (T, T*, RT); their botanical prototypes have been established. The arrangement of leaves in many plants is not described by the proposed apparatus (they optimize the light flux differently), which guides scientists to search for deeper patterns and methods of mathematical description.

Updating direct methods II. Reduction of the structural complexity when triplet invariants are estimated via the Patterson map

Direct methods have practically solved the phase problem for small–medium-size molecules but have substantially failed in macromolecular crystallography. They have two main limitations: a strong dependence on structural complexity and the need to work with atomic-resolution data. Many attempts have been made to broaden their field of applicability, for example the use of some a priori information to make the estimate of the triplet invariant phases more effective. Unfortunately none of these new approaches allowed the successful application of direct methods to proteins and nucleic acids. Direct methods are still a niche tool in macromolecular crystallography. In a recent publication [Giacovazzo (2019). Acta Cryst. A75, 142–157] the method of joint probability distributions has been modified to take into account new sources of prior information, one of which is relevant to this article: the Patterson map. In practice, it has been shown that with prior knowledge of the interatomic vectors one is able to modify the classic Cochran reliability parameter for estimating the triplet invariant phases. The article was essentially theoretical in nature, and no attempt was described to test the practical usefulness of the new probabilistic formulas. This work is therefore the first application of the new method. It is shown that the use of the Patterson map as prior information substantially improves the Cochran estimate of triplet phases; the phase error distribution for the new estimates, even if it is related to macromolecular structures, becomes similar to that obtained for medium-size structures. In some ways, it is as if the use of the Patterson information reduces the structural complexity, thus allowing a more general use of direct methods in macromolecular crystallography. Atomic resolution no longer seems to be a necessary ingredient for the applicability of direct methods; tests show that the apparent reduction in structural complexity also occurs in macromolecular structures with experimental data having a resolution of 2.3 Å. A number of test structures have been used to show the potential of the new technique.

Validation of electron-microscopy maps using solution small-angle X-ray scattering.

The determination of the atomic resolution structure of biomacromolecules isessential for understanding details of their function. Traditionally, such a structure determination has been performed with crystallographic or nuclear resonance methods, but during the last decade, cryogenic transmission electron microscopy (cryo-TEM) has become an equally important tool. As the blotting and flash-freezing of the samples can induce conformational changes, external validation tools are required to ensure that the vitrified samples are representative of the solution. Although many validation tools have already been developed, most of them rely on fully resolved atomic models, which prevents early screening of the cryo-TEM maps. Here, a novel and automated method forperforming such a validation utilizing small-angle X-ray scattering measurements, publicly available through the new software package AUSAXS, is introduced and implemented. The method has been tested on both simulated and experimental data, where it was shown to work remarkably well as a validation tool. The method provides a dummy atomic model derived from the EM map which best represents the solution structure.

Hydrogen bonding, halogen bonding and C–H…π interactions governing the supramolecular architecture of 1-(4-(4-bromophenyl)piperazin-1-yl)-2-chloroethan-1-one: Insights from X-ray crystallography, DFT calculations and urease inhibitory assessment

The use of organic ligands for the generation of supramolecular architectures exhibiting interesting structural properties in crystal engineering have garnered significant attention. In this context, the current work highlights the use of 1-(4-(4-bromophenyl)piperazin-1-yl)-2-chloroethan-1-one (3) as a valuable synthon for evaluating the conspicuous role of several types of noncovalent interactions governing the formation of supramolecular assemblies. Compound 3 was successfully synthesized in good yield using a facile two-step approach and characterized through spectroscopic and X-ray crystallographic methods. Noncovalent interactions including C—H…O, C—H…Cl, C—H…π as well as C—Br…Cl halogen bonds were found as the key contacts in the crystal packing of compound 3. DFT calculations were employed to identify and assess the impact of the C—Br…Cl halogen bond, utilizing MEP, QTAIM, and NBO analyses. These studies confirmed that the Br atom acts as the halogen bond donor and the Cl atom as the halogen bond acceptor. Assessment of urease inhibitory efficacy revealed that the tested compound exhibits a 16-folds superior inhibition than thiourea. Computational modeling analysis augmented the in vitro results by exhibiting numerous favorable binding interactions between compound 3 and active site residues such as TYR32, LYS709, PHE712, ASP730, VAL744 and MET746. The pharmacokinetic evaluation classified the investigated piperazine derivative into a druggable compound.

Crystal structure via fluctuation scattering.

Crystallography is a quintessential method for determining the atomic structure of crystals. The most common implementation of crystallography uses single crystals that must be of sufficient size, typically tens of micrometres or larger, depending on the complexity of the crystal structure. The emergence of serial data-collection methods in crystallography, particularly for time-resolved experiments, opens up opportunities to develop new routes to structure determination for nanocrystals and ensembles of crystals. Fluctuation X-ray scattering is a correlation-based approach for single-particle imaging from ensembles of identical particles, but has yet to be applied to crystal structure determination. Here, an iterative algorithm is presented that recovers crystal structure-factor intensities from fluctuation X-ray scattering correlations. The capabilities of this algorithm are demonstrated by recovering the structure of three small-molecule crystals and a protein crystal from simulated fluctuation X-ray scattering correlations. This method could facilitate the recovery of structure-factor intensities from crystals in serial crystallography experiments and relax sample requirements for crystallography experiments.

Flexible nonlinear photonics of heterostructured MXene-derived quantum dots

High-quality V2C–VO2-heterostructured MXene-derived quantum dots (MQDs) were hydrothermally fabricated and characterized using microscopic, crystallographic, and spectroscopic methods. Open-aperture Z-scans were employed to investigate the nonlinear optical absorption of the MQDs irradiated using picosecond pulses at 532 nm. The results showed that the heterostructure converted the nonlinear optical behavior from saturable absorption to reverse saturable absorption. In individual V2C MQDs, saturable absorption was ascribed to the bleaching of ground-state free electrons, while in heterostructured V2C–VO2 MQDs, reverse saturable absorption was derived from two-photon-induced free-carrier absorption. The nonlinear absorption coefficient of the V2C–VO2 heterostructured MQDs was comparable to those of previously reported MQDs. This research could provide insight into the integration of heterostructured MQDs and promote the advanced photonics of MQDs and their applications in optoelectronic devices.

Host-Guest Cocrystallization of Phenanthrene[2]arene Macrocycles Facilitating Structure Determination of Liquid Organic Molecules.

Single-crystal X-ray diffraction analysis has emerged as the most reliable method for determining the structures of organic molecules. However, numerous analytes, such as liquid organic molecules, pose challenges in crystallization, making their structures directly elusive via X-ray crystallography methods. Herein, we introduced the rapid cocrystallization of a macrocycle named phenanthrene[2]arene (PTA, host) with 15 liquid organic molecules (guests). The guest liquid organic molecules were successively cocrystallized with the aid of the PTA host. Moreover, the chemical structures of the liquid organic molecules could be determined through single-crystal X-ray diffraction analysis. PTA exhibited high adaptivity and was capable of encapsulating liquid organic molecules without forming covalent bonds or strong directional interactions. The results revealed that the adaptive crystals of PTA exhibited excellent cocrystallization capacity. Weak noncovalent interactions between the host and guest molecules were crucial for organizing the guests in an ordered pattern.

Nondestructive Single-Atom-Thick Crystallographic Scanner via Sticky-Note-Like van der Waals Assembling-Disassembling.

Crystallographic characteristics, including grain boundaries and crystallographic orientation of each grain, are crucial in defining the properties of two-dimensional materials (2DMs). To date, local microstructure analysis of 2DMs, which requires destructive and complex processes, is primarily used to identify unknown 2DM specimens, hindering the subsequent use of characterized samples. Here, a nondestructive large-area 2D crystallographic analytical method through sticky-note-like van der Waals (vdW) assembling-disassembling is presented. By the vdW assembling of veiled polycrystalline graphene (PCG) with a single-atom-thick single-crystalline graphene filter (SCG-filter), detailed crystallographic information of each grain in PCGs is visualized through a 2D Raman signal scan, which relies on the interlayer twist angle. The scanned PCGs are seamlessly separated from the SCG-filter using vdW disassembling, preserving their original condition. The remaining SCG-filter is then reused for additional crystallographic scans of other PCGs. It is believed that the methods can pave the way for advances in the crystallographic analysis of single-atom-thick materials, offering huge implications for the applications of 2DMs.

Five undescribed cyclopeptides from Cordyceps militaris

Ustiloxins I–M (1–5), five undescribed cyclopeptides bearing a 15-membered macrocyclic skeleton, were isolated from Cordyceps militaris. The structures of 1 and 5 were identified by spectroscopic and crystallographic methods, whereas the structures of 2–4 were assigned by spectroscopic and computational approaches. Biological evaluation of all the compounds toward human triple-negative breast cancer cells revealed that compounds 4 and 5 are toxic with IC50 values of 64.29 μM and 28.89 μM, respectively.

Hymoins A–C, three unusual polycyclic polyprenylated acylphloroglucinols with lipid-lowering activity from Hypericum monogynum

Hymoins A–C (1–3), three unusual polycyclic polyprenylated acylphloroglucinols (PPAPs) were isolated from the flowers of Hypericum monogynum. Hymoin A features the first intriguing 6/5/5/5/7 pentacyclic caged PPAP. Hymoin B is characterized by an unprecedented rearranged 5/6/8 tricyclic ring system, while hymoin C represents the first rearranged PPAP with a fantastic spirocyclic 5/6/7 ring system. Their structures were established by extensive spectroscopic analysis, X-ray crystallography, and computational methods. The plausible biosynthetic routes for the compounds were also proposed. In oleic acid (OA)-induced HepG2 cells, all compounds exhibited significant lipid-lowering activity at the concentrations of 2–8 μmol/L. Further mechanistic study implied that compound 1 exhibited excellent lipid-lowering activity in OA-induced HepG2 cells through inhibiting the proteins of free fatty acids synthesis and improving lipidolysis.

Abstract 6011: Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo

Abstract Tumors carrying defects in DNA mismatch repair (MMR-d) display high tumor mutational burden (TMB) and increased tumor neoantigen levels. MMR-d leads to accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences generating microsatellite instability (MSI). Cancers that harbor &amp;gt;40% microsatellite variations are described as MSI-high (MSI-H). These tumors display unique clinical features including better prognosis, increased immune infiltration and remarkable response to immune checkpoint blockade (ICB) therapy. Colorectal cancer (CRC) patients with MMR-d often respond to therapies based on immune checkpoint blockade, whilst those with mismatch repair proficient (MMR-p) tumors do not. Therefore, identifying strategies to enhance the efficacy of immunotherapeutic treatments and transform this prevalent subgroup of CRCs from immunologically cold to hot remains an urgent and an unmet clinical need. We discovered NP1867 a first-in-class, potent, selective, covalent small molecule inhibitor of the DNA Mismatch Repair protein PMS2. We show by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in mechanistic and functional cell-based assays of MMR-dependent DNA repair. Continuous treatment of murine CRC cell line CT26 (MMR-p) with NP1867 increases TMB, specifically enriches gained mutations with typical MMR-d related COSMIC mutational signatures, and elicits MSI-H status in a time-dependent manner, demonstrating for the first time that inhibition of PMS2 with a small molecule phenocopies the inactivation of MMR observed in patients. Remarkably, inoculation of immune-competent mice with CT26 cells pre-treated with NP1867 elicits tumor regressions in anti-PD1-treated animals, recapitulating the unique clinical features of MMR-d/MSI-H tumors. Conversely, no response is observed in immune-competent animals inoculated with control-treated cells. The extent of in vivo response correlates with the duration of in vitro NP1867 treatment and with the MSI-H status of treated CT26 cells. In conclusion, small molecule NP1867 functionally inhibits DNA MMR by targeting PMS2, enriches gained mutations with MMR-d COSMIC signatures, elicits MSI-H status, and enhances immune surveillance in preclinical models. These findings pave the way for the development of orally bioavailable compounds suitable for long-term dosing in animal models. Citation Format: Eleonora Piumatti, Alexia Hervieu, Philippe Riou, Julian Blagg, Adam Peall, Sam Weeks, Maria T. Rodríguez-Plata, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Paige Tongue, Tessa McLaren, Kalpesh Parmar, Pradip Patel, David Clark, Gareth Langley, Charles Nichols, Benoît Rousseau, Paul Winship, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6011.