Potent Inhibitor Research Articles

Unlocking potent inhibitors for Mycobacterium tuberculosis NaMN adenylyltransferase: a multi-database exploration through integrated structure and ligand-based pharmacophore modelling, dynamics and energetic analysis

ABSTRACT Ability of mycobacteria to develop resistance against new antibiotics is demanding novel screening methodologies beyond conventional methods for new anti-tubercular drugs. Enzymes of NAD biogenesis in mycobacteria present a viable therapeutic target for both latent and active tuberculosis. It has recently been demonstrated that NaMN adenylyltransferase is an essential enzyme for mycobacterial survival. We present here the pharmacophore-based virtual screening strategies to find potential inhibitors against the NaMN adenylyltransferase. Pharmacophores were designed using benzimidazolium derivatives which occupy a unique site inducing the formation of dimer of dimers of NaMN adenylyltransferase and blocking its activity. Several such compounds exhibited high affinity binding to the NaMN adenylyltransferase structure, distinguishing them as high-scoring inhibitors. Further, molecular dynamics simulations along with MM-PBSA calculations were performed to evaluate them. Notably, ZINC34698873 from ligand-based pharmacophore modelling and CCG30319 from structure-based pharmacophore modelling both showed substantial binding and inhibitory effects against this enzyme. Simulation results indicate that these possible inhibitors were able to effectively adopt and form stable complexes within the binding pocket. Furthermore, the assertion that these molecules create strong contacts with dimer interface region residues were further supported by the low binding energy obtained from MM-PBSA. HIGHLIGHTS Virtual screening for new antitubercular medicines that targets NaMN adenylyltransferase. Ligand and structure-based pharmacophore modelling was employed in the study. The binding efficacy is validated by MM-PBSA calculations and molecular dynamics simulations. Compounds ZINC34698873 and CCG30319, demonstrating strong binding affinity to NaMN adenylyltransferase, show promise as candidates for antitubercular drugs.

Design, synthesis, biological assessments and computational studies of 3-substituted phenyl quinazolinone derivatives as promising anti-cancer agents

A new series of 3-substituted phenyl quinazolinone derivatives were designed and synthesized as anti-cancer agents. The most potent derivative with IC50 values of 12.84 ± 0.84 and 10.90 ± 0.84 µM against MCF-7 and SW480 cell lines was comparable to Cisplatin and Erlotinib as positive controls. Cell cycle analysis showed that the most active compound could arrest at S phase in MCF-7 breast cancer cells. The apoptosis assay demonstrated the induction of apoptosis in the MCF-7 cell line, too. Molecular docking results showed better accommodation of the most active compound through hydrogen bonding interaction in the binding site of EGFR enzyme. Molecular dynamics simulations for the potent analogue demonstrated well binding stability compared to the less active analogue, with a lower RMSD, Rg and more interactions with the original active site residues. DFT calculations were performed on the active and inactive compounds, using Gaussian 09 at the M06-2X/6–31 + G(d) theoretical level. ADME (Absorption, Distribution, Metabolism, and Excretion) properties showed that most of the compounds are in acceptable range of Lipiniski rule. These findings underscore the potential of the synthesized compounds as potent cytotoxic inhibitors and provide insights for developing effective treatments for cancer therapy.

Identification of a RANKL/TNF-α Dual-Inhibitor as a Potential Disease-Modifying Agent for the Treatment of Knee Osteoarthritis.

Osteoarthritis (OA) is a multifactorial degenerative disease involved subchondral bone remodeling, cartilage destruction and synovium inflammation. While receptor activator of nuclear factor-κB ligand (RANKL), a tumor necrosis factor (TNF) superfamily protein, is the critical regulator in bone metabolism associated with subchondral bone resorption, TNF-α is also an important inflammatory factor involved in the OA inflammation and cartilage destruction. Based on previous compound Y1599, we identified a novel tetrahydro-β-carboline derivative Y2641 with both RANKL and TNF-α inhibition in this study. Y2641 exhibited potent RANKL-induced osteoclastogenic inhibition (IC50 = 109.1 nM), and had anti-inflammatory and cartilage destruction inhibiting effects at 10 μM with low cytotoxicity. SPR assays demonstrated the binding affinity of Y2641 to RANKL (Kd = 3.984 μM) and TNF-α (Kd = 18.59 μM). In vivo assay further revealed the disease-modifying effects of Y2641 in OA rats, establishing Y2641 as a promising lead compound for the development of disease-modifying osteoarthritis drugs.

Species-Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with In Vivo Antialphaviral Activity.

RA-0002034 (1) is a potent covalent inhibitor targeting the nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species, such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing chikungunya (CHIKV) viral loads in multiple tissues. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a potential antialphaviral therapeutic to further define the contribution of hepatic and non-hepatic GST metabolism to its clearance in humans.

Discovery of a Chiral 2,4-Substituted Pyrrolo[2,3-d]pyrimidine as a Potent, Selective, and Orally Bioavailable LRRK2 Inhibitor.

Inhibition of leucine-rich repeat kinase (LRRK2) activity with small molecules has emerged as a potential novel therapeutic target for Parkinson's disease (PD). We have previously reported the identification of SRI-29132 as a potent LRRK2 inhibitor, but the presence of a 6-thioether moiety, which is an oxidative liability, precludes its further development. Herein, we report another hit-to-lead optimization study that led to the discovery of the chiral 2,4-substituted pyrrolo[2,3-d]pyrimidine series as potent LRRK2 inhibitors. Our lead analog 6, derived from a high-throughput screening hit SRI-31255, exhibits excellent LRRK2 inhibition activity and, high selectivity across the kinome. Further, the molecule has acceptable absorption, distribution, metabolism, and excretion (ADME), and pharmacokinetic (PK) properties, as well as brain permeability and no off-target liabilities. This new class of compounds serves as a novel series for further study in the development of LRRK2 inhibitors for therapy.

Discovering the Cholinesterase Inhibitory Potential of Thiosemicarbazone Derivatives through In vitro, Molecular Docking, Kinetics, and Dynamics Studies.

The current study explored the cholinesterase inhibitory activities of some thiosemicarbazone derivatives bearing 2,4-dichloro phenylacetic acid scaffold. This study aimed to screen the synthesized derivatives for their in vitro acetylcholine and butyrylcholinesterase inhibition. These compounds were synthesized by refluxing 2,4-dichloro phenylacetic acid with sulfuric acid in ethanol to get the ester, which was further refluxed with thiosemicarbazide in ethanol to get the desired compound (2). Different benzaldehydes were treated with compound (2) in ethanol having a catalytic amount of acetic acid to get thiosemicarbazones. In the series, seven compounds, including compounds 2c, 2a, 2b, 2d, 2g, 2e, and 2f, displayed excellent acetylcholinesterase inhibition activities in the range of IC50 values from 41.51 ± 3.88 to 95.48 ± 0.70 μM, surpassing than the standard galantamine (IC50 = 104.5 ± 1.20 μM). Also, compounds 2a, 2g, 2h, 2f, 2b, and 2d with IC50 values ranging from 64.47 ± 2.74 to 80.62 ± 0.73 μM exhibited potent inhibition against butyrylcholinesterase enzyme, being similar to the standard galantamine (IC50 = 156.8 ± 1.50 μM). The molecular docking investigation was performed to assess the binding affinity of the compounds with the active site of the enzyme. These compounds, along with the docked complexes, specifically AChE-compound 2a and BuChE-compound 2g, were chosen and subjected to 100-nanosecond molecular dynamics simulations. The simulations demonstrated strong stability of the ligands within the active pockets of AChE and BuChE enzymes. These derivatives exhibited superior acetylcholinesterase and butyrylcholinesterase inhibitory activities compared to galantamine, with molecular docking and dynamic simulations confirming their strong binding affinity with the active sites of the enzymes.

Intracellular and Extracellular Efficacy of Homoisoflavone Derivatives Against Mycobacterium tuberculosis: Progress Toward Novel Antitubercular Agents.

Tuberculosis (TB) is the leading cause of death among infectious diseases, and new studies focusing new anti-TB drugs are urgent due to the emergence of drug-resistant strains. In this regard, homoisoflavones, anunderexplored subgroup of flavonoids, were explored for their potential as anti-TB agents. A series of 42 derivatives was synthesized, and assessed for their ability to inhibit the growth of Mycobacterium tuberculosis (Mtb). Derivatives 19, 22, and 41 showed good selectivity index and potent inhibition of the Mtb H37Rv(MIC90 2.2, 3.8, and 1.9 µM, respectively), while derivatives 22 and 41 stood out for inhibiting the hypervirulent clinical isolate Mtb M299 (MIC90 1.5 and 2.5 µM, respectively), surpassing the potency of rifampicin. Moreover, these derivatives inhibited the growth of Mtb H37Rv in infected macrophages, where derivative 41, featuring the nitrofuranyl group as B-ring and the piperidine group at the 7-position of A-ring, emerged as the most potent derivative (IC50 5.2 µM). Using calculated steric and electronic structural descriptors, an unprecedented structure-activity relationshipfor the homoisoflavone scaffold was established, and in silico studies also indicated good oral bioavailability and low toxicity for thesecompounds. These findings underscore the potential of homoisoflavone derivatives as candidates for further development as anti-TB agents.

Molecular Basis of Pseudomonas syringae pv actinidiae Levansucrase Inhibition by a Multivalent Iminosugar.

Levansucrases are a class of polysaccharide-processing enzymes widely distributed among plant pathogenic bacteria, such as Pseudomonas syringae and Erwinia amylovora. Therefore, the modulation of levansucrase activity could represent a new strategy to reduce the microbial survival of such bacteria. Herein, we identified a tetravalent pyrrolidine iminosugar (TPIS) as the first levansucrase inhibitor described to date. TPIS reversibly inhibits sucrose hydrolysis and levan polymerization of levansucrase derived from different bacterial genotypes of P. syringae, showing competitive behavior and an inhibition constant (Ki) in the micromolar range. Interestingly, the monovalent pyrrolidine iminosugar (PIS) analogue shows negligible inhibition, suggesting that multivalency plays a pivotal role in the interaction with levansucrase. To gain insight into the binding mechanism, the X-ray crystal structures of the beta levansucrase isoform from P. syringae pv actinidiae (Psa) in its native form and in complex with TPIS were solved, confirming TPIS as a competitive inhibitor of levansucrases. Only a portion of TPIS, corresponding to one chain of the tetravalent iminosugar derivative, was visible in the electron density maps. Nevertheless, our structural data provided an adequate comprehension of the inhibitor/enzyme interactions, sufficient to exclude some of the possible inhibition mechanisms justifying a multivalent effect and pave the way for the development of new, more potent inhibitors.

Structural insights of WBmDapE and deciphering of potent anti-filarial inhibitors: a state-of-art computational approach.

Lymphatic filariasis (LF) stands as a debilitating tropical ailment, impacting a considerable global populace. Existing drug therapies for LF exhibit limited effectiveness across all parasite stages, thereby accentuating the imperative for novel anti-filarial medications characterized by enhanced prognostic attributes and minimized adverse reactions. A promising avenue involves targeting the microbial enzyme WBmDapE, pivotal in worm survival and intricately linked to the lysine biosynthetic pathway and peptidoglycan cell wall construction. This investigation employs in silico methodologies encompassing molecular docking, Molecular Dynamics Simulation (MDS), conformational analysis, Shape-Based Virtual Screening (SBVS), ADMETox, MMGBSA, and Density Functional Theory (DFT) calculations to discern potential inhibitors of WBmDapE. Through discerning the conformational shifts of the WBmDapE-bound substrate and product, key amino acids implicated in substrate binding (Arg182 and Asp248) are unveiled. While the apo and substrate-bound structures exhibit an open conformation, the product-bound structure displays marked conformational alterations, including shifts within the catalytic domain and the cofactor in the dimerization domain, suggestive of an active and closed conformation. From the prism of shape-based virtual screening, two preeminent compounds (ZINC42784280 and ZINC84308049) have surfaced as potential hits. These compounds evince heightened binding affinity, optimal binding free energy, pivotal hydrogen bond interactions, and akin attributes to the product-bound complex. Subsequently, these compounds emerge as prospective candidates for filariasis treatment. In summation, our study furnishes invaluable insights into the fabrication of innovative WBmDapE inhibitors, potentially serving as anti-filarial agents. Rigorous experimental substantiation and fine-tuning of these compounds are requisite for prospective therapeutic interventions against LF.

SGLT2 Inhibition by Enavogliflozin Significantly Reduces Aβ Pathology and Restores Cognitive Function via Upregulation of Microglial AMPK Signaling in 5XFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline. Metabolic dysfunctions, particularly type 2 diabetes mellitus (T2DM), have been implicated in AD pathogenesis, highlighting the potential for novel therapeutic approaches targeting shared underlying mechanisms. Here, we investigate sodium-glucose cotransporter 2 (SGLT2) inhibition as a therapeutic strategy for AD using Enavogliflozin, a potent SGLT2 inhibitor, in the 5XFAD mouse model. Five-month-old 5XFAD mice were treated with Enavogliflozin (0.1 or 1 mg/kg) or vehicle for 8 weeks. The higher dose significantly improved cognitive performance in Y-maze and Morris Water Maze tests, which correlated with enhanced synaptic plasticity and increased acetylcholine levels. Moreover, Enavogliflozin treatment reduced Aβ pathology and plaque burden, particularly affecting larger plaques. Mechanistically, SGLT2 inhibition attenuated neuroinflammation by suppressing NF-κB signaling and proinflammatory cytokine production while promoting microglial recruitment to plaques. Invitro and exvivo analyses further revealed that Enavogliflozin enhances microglial phagocytic capacity via AMPK-mediated mitochondrial biogenesis and function. These findings highlight the multifaceted neuroprotective effects of SGLT2 inhibition in AD, demonstrating its potential to mitigate pathology and improve cognitive function. By uncovering its impact on neuroinflammation and microglial function, this study establishes SGLT2 inhibition as a promising therapeutic avenue for AD and other neurodegenerative disorders.

The inhibition of monoamine oxidase by 2-methylbenzo[d]oxazole derivatives

Abstract The monoamine oxidase (MAO) enzymes metabolise neurotransmitter amines and are drug targets for the treatment of neuropsychiatric and neurodegenerative disorders. Over the past several decades, MAO inhibitors have been used as antidepressants and antiparkinsonian agents. The present study investigated the MAO inhibition properties of a series of benzoxazole derivatives. Many benzoxazole-containing drugs have been marketed and are used for the treatment of a wide variety of conditions. Thirteen 2-methylbenzo[d]oxazole derivatives (1a–f, 2a–g) were synthesised and evaluated as in vitro inhibitors of human MAO. The results showed that the benzoxazole derivatives were potent MAO inhibitors. Compounds 1d and 2e were the most potent MAO-B inhibitors with IC50 values of 0.0023 and 0.0033 µM, respectively. The most potent MAO-A inhibition was displayed by compounds 2c and 2e with IC50 values of 0.670 and 0.592 µM, respectively. It may be concluded that the benzoxazole derivatives of this study could be useful lead compounds for the development of clinically useful MAO inhibitors.

Biosynthesis of the fungal cyclic lipodepsipeptide pleosporacin, a new selective inhibitor of the phytopathogen Botrytis cinerea.

Bioactivity-guided isolation led to the identification of the cyclic lipodepsipeptide pleosporacin (1) from the mycelia extract of fungal strain Pleosporales sp. IBWF 020-21, a potent selective inhibitor of the fungal phytopathogen Botrytiscinerea. The structure and stereochemistry of 1 were elucidated by NMR and Marfey analysis, respectively. Genome mining identified a candidate biosynthetic gene cluster encoding a hexamodular NRPS, PleA, a fatty acyl-AMP ligase, PleB, and an aspartate decarboxylase, PleC. Reconstitution of pleABC allowed for heterologous production of 1 in Aspergillusoryzae and confirmed the identity of the ple cluster. Based on our findings we propose a biosynthetic route, with PleB catalyzing lipoinitiation and PleC providing the non-proteinogenic amino acid β-alanine for the assembly of 1.

Enzymatic combinatorial synthesis of E-64 and related cysteine protease inhibitors.

E-64 is an irreversible cysteine protease inhibitor prominently used in chemical biology and drug discovery. Here we uncover a nonribosomal peptide synthetase-independent biosynthetic pathway for E-64, which is widely conserved in fungi. The pathway starts with epoxidation of fumaric acid to the warhead (2S,3S)-trans-epoxysuccinic acid with an Fe(II)/α-ketoglutarate-dependent oxygenase, followed by successive condensation with an L-amino acid by an adenosine triphosphate grasp enzyme and with an amine by the fungal example of amide bond synthetase. Both amide bond-forming enzymes display notable biocatalytic potential, including scalability, stereoselectivity toward the warhead and broader substrate scopes in forming the amide bonds. Biocatalytic cascade with these amide bond-forming enzymes generated a library of cysteine protease inhibitors, leading to more potent cathepsin inhibitors. Additionally, one-pot reactions enabled the preparative synthesis of clinically relevant inhibitors. Our work highlights the importance of biosynthetic investigation for enzyme discovery and the potential of amide bond-forming enzymes in synthesizing small-molecule libraries.

Advances in Gastroesophageal Reflux Disease Management: Exploring the Role of Potassium-Competitive Acid Blockers and Novel Therapies

Gastroesophageal reflux disease (GERD) is a prevalent chronic gastrointestinal disorder that affects a substantial proportion of the global population. It is characterized by the extensive backward flow of stomach contents into the esophagus, leading to troublesome symptoms and potential complications. Proton pump inhibitors (PPIs) have long been the cornerstone of pharmacological treatment for GERD, effectively suppressing gastric acid secretion. However, a substantial subset of patients, referred to as PPI-refractory GERD, experience inadequate symptom control despite optimal PPI therapy. GERD significantly impacts patients’ quality of life, affecting domains, such as vitality, pain, and physical functioning. Consequently, there is an urgent need for alternative therapeutic strategies and novel pharmacologic agents to provide more effective, long-term relief. Emerging treatment options include potassium-competitive acid blockers (PCABs) like vonoprazan, which offer more potent and sustained inhibition of gastric acid secretion compared to traditional PPIs. Additionally, prokinetic agents such as itopride have gained attention due to their potential to improve GERD symptoms by enhancing gastrointestinal motility and accelerating gastric emptying. This article reviews the mechanisms of action, clinical efficacy, and potential of these novel therapeutic approaches in improving patient outcomes in GERD management. With the growing prevalence of PPI resistance and side effects, a personalized, multifaceted approach to treatment is becoming increasingly necessary to optimize care for patients with GERD.

Addition of Lithiated 1,3-Dithiane and Nitronate to Sugar-Derived Imines: Synthesis of Homoiminosugars and Pipecolic Acid Analogues.

The one-pot two-step stereoselective synthesis of 1,2-trans-1-C-1,3-dithian-2-yl and 1,2-cis-1-C-nitromethyl iminosugars from sugar lactams is disclosed, exploiting Schwartz's reagent triggered amide to imine reduction followed by Corey-Seebach or nitro-Mannich-mediated functionalizations. Processing of the dithiane moiety provided the naturally occurring β-HNJ and α-HMJ homoiminosugars, while processing of the nitromethyl group gave access to α-HNJ and pipecolic acid derivatives. Epimerization of this function was also successfully examined. Final hydrogenolysis furnished a set of known and new glycomimetics, amongst which two compounds displayed potent glycosidase inhibition.

Suppressing chondrocyte cuproptosis by syringaresinol-4-O-β-d-glucoside alleviates gouty arthritis

BackgroundGouty arthritis is a rheumatic disease characterized by synovial inflammation and cartilage damage. Current therapeutic options for gouty arthritis, such as colchicine, primarily relieve the symptoms, which makes treatment challenging.MethodsWe employed an in vitro co-culture system of chondrocytes and macrophages to simulate gouty arthritis and screen compounds that can inhibit monosodium urate (MSU) associated macrophage inflammation and chondrocytes degeneration. We further elucidated the cuproptosis mechanism in chondrocytes by qPCR and Western blotting analyses. Both acute and chronic gouty arthritis mouse models were established to evaluate the therapeutic efficacy of candidate drugs against gouty arthritis.ResultsMSU upregulates the expression of inflammatory cytokines in macrophages and simultaneously induces cuproptosis in chondrocytes. By screening 24 compounds, we identified syringaresinol-4-O-β-d-glucoside (SSG), a furanoid lignan, as a potent inhibitor of macrophage-mediated inflammation and chondrocyte cuproptosis. Mechanistically, SSG inhibited MSU-induced activation of the NF-κB and NLRP3 pathways in macrophages. Furthermore, SSG regulated the expression of sulfur-linked mitochondrial enzymes (e.g., DLAT) in the cuproptosis pathway, thereby inhibiting the upstream regulator FDX1 in chondrocytes. SSG not only alleviated inflammatory pain but also protected against cartilage damage and improved motor dysfunction in the mice models of acute and chronic gouty arthritis.ConclusionSSG can serve as a promising therapeutic option for gouty arthritis in clinical settings by suppressing inflammation and preserving cartilage integrity.

Steroidogenesis and CYP17A1 inhibition: development of potent inhibitors for adrenal and gonadal steroid biosynthesis

Steroidogenesis and CYP17A1 inhibition: development of potent inhibitors for adrenal and gonadal steroid biosynthesis

Kaurenoic acid is a potent inhibitor of SARS-CoV-2 RNA synthesis, virion assembly, and release in vitro

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 (COVID-19) pandemic, continues to pose global health challenges despite the availability of approved vaccines and antiviral drugs. The emergence of new variants of SARS-CoV-2 and ongoing post-COVID complications necessitate continuous exploration of effective treatments. Kaurenoic acid (KA) is a tetracyclic diterpenoid isolated from plants of the Copaifera genus and has been previously recognized for its anti-inflammatory, antibacterial, antifungal, and antitumor properties. However, there is a lack of knowledge about the in vitro effects of KA on viruses. Here, we evaluated its effect on SARS-CoV-2 replication for the first time.Methods and ResultsKA demonstrated a high selective index of 16.1 against SARS-CoV-2 and robust effectiveness against the B.1.617.2 (Delta) and BA.2 (Omicron) variants. Mechanistically, KA was shown to impair the post-entry steps of viral replication. In a subgenomic replicon system, we observed a decrease in viral RNA synthesis in different cell lines. Using an infectious virus, a larger reduction in the release of SARS-CoV-2 virions was observed. We suggest that KA interacts with SARS-CoV-2 proteases through molecular docking.ConclusionIn conclusion, KA emerges as an inhibitor of SARS-CoV-2 proteases and, consequently, its replication cycle. It could be a good candidate for further investigation in clinical assays against SARS-CoV-2 infection.

Discovery of Highly Potent and Selective EZH2 Covalent Inhibitors via Incorporating Basic Amines.

Targeted covalent inhibition is a promising strategy to address the high dose and acquired drug resistance issues of the first-generation EZH2 noncovalent inhibitors. Recently we have reported a new generation of highly potent EZH2 covalent inhibitors, but further optimization to enhance aqueous solubility is required. Here, we described the systematic optimization of EPZ-6438 by preserving the aqueous groups, resulting in the identification of a highly potent and selective EZH2 covalent inhibitor 13, which displayed nanomolar potency in biochemical and cellular assays. Moreover, SAM competition experiments preliminarily confirmed that 13 was noncompetitive with SAM, leading to the remarkable reduction of the H3K27Me3 marker. In addition, 13 exhibited superior cell growth inhibition in the EZH2 mutant cancer cell lines. The discovery of 13 holds promise for the development of highly potent EZH2 covalent inhibitors.

Design, Synthesis, Anticancer Evaluation and Molecular Docking of Pyrimidine, Pyrido[4,3-d]pyrimidine and 5,6,7,8-Tetrahydropyrido[3,4-d]pyrimidine Derivatives as Novel KRAS-G12D Inhibitors and PROTACs

Background: KRAS-G12D mutations drive 20–50% of pancreatic/biliary cancers yet remain challenging to target due to GTP-pocket conservation and high cellular GTP levels. While allosteric inhibitors targeting the SWII pocket (e.g., MRTX1133) show promise, limited chemical diversity and paradoxical cellular/enzymatic activity relationships necessitate the exploration of novel scaffolds. This study aims to develop KRAS-G12D inhibitors and PROTACs to offer a selection of new chemical entities through systematic structure–activity optimization and evaluate their therapeutic potential through PROTAC derivatization. Methods: Eleven compounds featuring heterocyclic cores (pyrimidine/pyrido[4,3-d]pyrimidine/5,6,7,8-tetrahydroprodo[3,4-d]pyrimidine) were designed via structure-based drug design. Antiproliferative activity against KRAS-G12D (Panc1), KRAS-G13D (HCT116) and wild-type (A549) cells was assessed using the CCK-8 assay. KRAS-G12D enzymatic inhibition was measured using a GTPase activity assay. Molecular docking simulations (Sybyl 2.0; PDB:7RPZ) elucidated binding modes. Two PROTACs were synthesized from lead compounds by conjugating E3 ligase linkers. All the novel inhibitors and PROTACs were characterized by means of NMR or HRMS. Results: Compound 10c demonstrated selective anti-proliferation in Panc1 cells (IC50 = 1.40 μM) with 4.9-fold greater selectivity over wild-type cells, despite weak enzymatic inhibition (IC50 > 10 μM). Docking revealed critical hydrogen bonds between its protonated 3,8-diazabicyclo[3.2.1]octane moiety and Asp12/Gly60. The enzymatic inhibitor 10k showed potent KRAS-G12D inhibition (IC50 = 0.009 μM) through homopiperazine-mediated interactions with Glu92/His95. Derived PROTACs 26a/b exhibited reduced potency (IC50 = 3–5 μM vs. parental 10k: 2.22 μM), potentially due to impaired membrane permeability. Conclusions: Eleven novel KRAS-G12D inhibitors with a seven-membered ring pharmacophore were synthesized. Compound 10c showed strong anti-proliferative activity, while 10k exhibited potent enzymatic inhibition. Two PROTACs were designed but showed no clear advantage over 10k. This study provides valuable insights for KRAS-targeted drug development.