Clinical Application of Individualized 3D-Printed Chest Wall Conformal Device in IMRT for Post-Mastectomy Breast Cancer.

Background Breast cancer is the most common malignant tumour in women. Radical mastectomy with postoperative radiotherapy is now the standard treatment for locally advanced breast cancer. Intensity-modulated radiotherapy (IMRT) has now been developed, which employs linear accelerators to deliver precise radiation to a tumour while minimizing the dose to surrounding normal tissue. It significantly improves the efficacy of breast cancer treatment. However, there are still some flaws that must be addressed. Objective To assess the clinical application of the three-dimensional (3D)-printed chest wall conformal device for breast cancer patients who need to be treated by chest wall intensity modulated radiotherapy (IMRT) after radical mastectomy. Methods The 24 patients were divided into three groups. During a computed tomography (CT) scan, patients in the study group were fixed by a 3D-printed chest wall conformal device, nothing in control group A, and a traditional 1-cm thick silica gel compensatory pad on the chest wall in control group B. The parameters of mean Dmax, Dmean, D2%, D50%, D98%, the conformity index (CI), and the homogeneity index (HI) of the planning target volume (PTV) are compared. Results The study group had the best dose uniformity (HI = 0.092) and the highest conformation (CI = 0.97), the worst in control group A (HI = 0.304, CI = 0.84). The mean Dmax, Dmean, and D2% of the study group were lower than control groups A and B (p<0.05). The mean D50% was higher than control group B (p<0.05), while the mean D98% was higher than control groups A and B (p<0.05). The mean Dmax, Dmean, D2%, and HI of control group A were higher than control group B (p<0.05), whereas the mean D98% and CI were lower than control group B (p<0.05). Conclusion By improving the efficacy of postoperative radiotherapy for breast cancer, using 3D-printed chest wall conformal devices may greatly improve the accuracy of repeating position fixation, increase the dose on the skin surface of the chest wall, optimise the dose distribution of the target area, and thus further reduce tumour recurrence and prolong patients' survival.

New ZW4864 Derivatives as Small-Molecule Inhibitors for the ß-Catenin/BCL9 Protein-Protein Interaction.

A series of 1-(3-(2-amino-2-oxoethoxy)phenyl)piperidine-3-carboxamide derivatives was reported as new small-molecule ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) inhibitors. Compounds 17-21 were discovered to inhibit the ß-catenin/BCL9 PPI with K i = 0.85-2.7 µM. The effects of 21 on the ß-catenin/BCL9 PPI in cellular context were demonstrated by ß-catenin/BCL9 pull-down inhibition and dose-dependent suppression of Wnt/ß-catenin signal transactivation. Notably, compound 21 is more potent than ZW4864, a previously reported analogue, in modulating transcription and expression of ß-catenin target genes and suppressing survival of ß-catenin-dependent cancer cells. The cellular on-target efficacy of 21 was demonstrated by ß-catenin rescue experiments. Compound 21 represents a promising starting point for further optimization of ß-catenin/BCL9 PPI inhibitors.

Discovery of an Orally Bioavailable Small-Molecule Inhibitor for the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Aberrant activation of Wnt/ß-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, ß-catenin, is a biologically rational approach to abolish hyperactivation of ß-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with ß-catenin and selectively disrupts the protein-protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and ß-catenin while sparing the ß-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses ß-catenin signaling activation, downregulates oncogenic ß-catenin target genes, and abrogates invasiveness of ß-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses ß-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore ß-catenin-related biology and a drug-like small-molecule ß-catenin/BCL9 disruptor for future drug development.

Discovery of 1-Benzoyl 4-Phenoxypiperidines as Small-Molecule Inhibitors of the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Structure-based design and optimization were performed to develop small-molecule ß-catenin/B-cell lymphoma 9 (BCL9) inhibitors and improve their inhibitory activities. Compound ZL3138 with a novel 1-benzoyl 4-phenoxypiperidine scaffold was discovered to disrupt the ß-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.96 µM in AlphaScreen competitive inhibition assays and displayed good selectivity for ß-catenin/BCL9 over ß-catenin/E-cadherin PPIs. The binding mode of new inhibitors was characterized by structure-activity relationship and site-directed mutagenesis studies. Protein pull-down assays indicate that this series of compounds directly binds with ß-catenin. Cellular target engagement and co-immunoprecipitation experiments demonstrate that ZL3138 binds with ß-catenin and disrupts the ß-catenin/BCL9 interaction without affecting the ß-catenin/E-cadherin interaction in living cells. Further cell-based studies show that ZL3138 selectively suppresses transactivation of Wnt/ß-catenin signaling, regulates transcription and expression of Wnt target genes, and inhibits the growth of Wnt/ß-catenin-dependent cancer cells.

Discovery of 2-(3-(3-Carbamoylpiperidin-1-yl)phenoxy)acetic Acid Derivatives as Novel Small-Molecule Inhibitors of the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for the suppression of hyperactive Wnt/ß-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we describe the medicinal chemistry optimization of a screening hit to yield novel small-molecule inhibitors of the ß-catenin/BCL9 interaction. The best compound 30 can disrupt the ß-catenin/BCL9 interaction with a Ki of 3.6 µM in AlphaScreen competitive inhibition assays. Cell-based experiments revealed that 30 selectively disrupted the ß-catenin/BCL9 PPI, while leaving the ß-catenin/E-cadherin PPI unaffected, dose-dependently suppressed Wnt signaling transactivation, downregulated oncogenic Wnt target gene expression, and on-target selectively inhibited the growth of cancer cells harboring aberrant Wnt signaling. This compound with a new chemotype can serve as a lead compound for further optimization of inhibitors for ß-catenin/BCL9 PPI.

Preclinical evaluation of [225Ac]Ac-DOTA-TATE for treatment of lung neuroendocrine neoplasms.

PURPOSE: There is significant interest in the development of targeted alpha-particle therapies (TATs) for treatment of solid tumors. The metal chelator-peptide conjugate, DOTA-TATE, loaded with the ß-particle emitting radionuclide 177Lu ([177Lu]Lu-DOTA-TATE) is now standard care for neuroendocrine tumors that express the somatostatin receptor 2 (SSTR2) target. A recent clinical study demonstrated efficacy of the corresponding [225Ac]Ac-DOTA-TATE in patients that were refractory to [177Lu]Lu-DOTA-TATE. Herein, we report the radiosynthesis, toxicity, biodistribution (BD), radiation dosimetry (RD), and efficacy of [225Ac]Ac-DOTA-TATE in small animal models of lung neuroendocrine neoplasms (NENs). METHODS: [225Ac]Ac-DOTA-TATE was synthesized and characterized for radiochemical yield, purity and stability. Non-tumor-bearing BALB/c mice were tested for toxicity and BD. Efficacy was determined by single intravenous injection of [225Ac]Ac-DOTA-TATE into SCID mice-bearing human SSTR2 positive H727 and H69 lung NENs. RD was calculated using the BD data. RESULTS: [225Ac]Ac-DOTA-TATE was synthesized with 98% yield, 99.8% purity, and displayed 97% stability after 2 days incubation in human serum at 37 °C. All animals in the toxicity study appeared healthy 5 months post injection with no indications of toxicity, except that animals that received ≥111 kBq of [225Ac]Ac-DOTA-TATE had chronic progressive nephropathy. BD studies revealed that the primary route of elimination is by the renal route. RD calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. For both tumor models, a significant tumor growth delay and time to experimental endpoint were observed following a single administration of [225Ac]Ac-DOTA-TATE relative to controls. CONCLUSIONS: These results suggest significant potential for the clinical translation of [225Ac]Ac-DOTA-TATE for lung NENs.

Direct targeting of ß-catenin in the Wnt signaling pathway: Current progress and perspectives.

Aberrant activation of the Wnt/ß-catenin signaling circuit is associated with cancer recurrence and relapse, cancer invasion and metastasis, and cancer immune evasion. Direct targeting of ß-catenin, the central hub in this signaling pathway, is a promising strategy to suppress the hyperactive ß-catenin signaling but has proven to be highly challenging. Substantial efforts have been made to discover compounds that bind with ß-catenin, block ß-catenin-mediated protein-protein interactions, and suppress ß-catenin signaling. Herein, we characterize potential small-molecule binding sites in ß-catenin, summarize bioactive small molecules that directly target ß-catenin, and review structure-based inhibitor optimization, structure-activity relationship, and biological activities of reported inhibitors. This knowledge will benefit future inhibitor development and ß-catenin-related drug discovery.

Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity.

Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC50 = 180 nM) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

Inhibition of ß-catenin/B cell lymphoma 9 protein-protein interaction using α-helix-mimicking sulfono-γ-AApeptide inhibitors.

The rational design of α-helix-mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein-protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting ß-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt ß-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with ß-catenin and disrupt ß-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein-protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the ß-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein-protein interactions.

Optimization of Peptidomimetics as Selective Inhibitors for the ß-Catenin/T-Cell Factor Protein-Protein Interaction.

The ß-catenin/T-cell factor (Tcf) protein-protein interaction (PPI) plays a critical role in the ß-catenin signaling pathway which is hyperactivated in many cancers and fibroses. Based on compound 1, which was designed to target the Tcf4 G13ANDE17 binding site of ß-catenin, extensive structure-activity relationship studies have been conducted. As a result, compounds 53 and 57 were found to disrupt the ß-catenin/Tcf PPI with the Ki values of 0.64 and 0.44 µM, respectively, and exhibit good selectivity for ß-catenin/Tcf over ß-catenin/E-cadherin and ß-catenin/adenomatous polyposis coli (APC) PPIs. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) cell viability assays revealed that 56, the ethyl ester of 53, was more potent than 53 in inhibiting viability of most of the Wnt/ß-catenin hyperactive cancer cells. Further cell-based studies indicated that 56 disrupted the ß-catenin/Tcf PPI without affecting the ß-catenin/E-cadherin and ß-catenin/APC PPIs, suppressed transactivation of Wnt/ß-catenin signaling in dose-dependent manners, and inhibited migration and invasiveness of Wnt/ß-catenin-dependent cancer cells.

AKR1B10 activates diacylglycerol (DAG) second messenger in breast cancer cells.

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/ßII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

Structure-Based Optimization of Small-Molecule Inhibitors for the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Structure-based optimization was conducted to improve the potency, selectivity, and cell-based activities of ß-catenin/B-cell lymphoma 9 (BCL9) inhibitors based on the 4'-fluoro- N-phenyl-[1,1'-biphenyl]-3-carboxamide scaffold, which was designed to mimic the side chains of the hydrophobic α-helical hot spots at positions i, i + 3, and i + 7. Compound 29 was found to disrupt the ß-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.47 µM and >1900-fold selectivity for ß-catenin/BCL9 over ß-catenin/E-cadherin PPIs. The proposed binding mode of new inhibitors was consistent with the results of site-directed mutagenesis and structure-activity relationship studies. Cell-based studies indicated that 29 disrupted the ß-catenin/BCL9 interaction without affecting the ß-catenin/E-cadherin interaction, selectively suppressed transactivation of Wnt/ß-catenin signaling, downregulated expression of Wnt target genes, and inhibited viability of Wnt/ß-catenin-dependent cancer cells in dose-dependent manners. A comparison of the biochemical and cell-based assay results offered the directions for future inhibitor optimization.

A Versatile Method to Determine the Cellular Bioavailability of Small-Molecule Inhibitors.

The determination of the cellular bioavailability of small-molecule inhibitors is a critical step for interpreting cell-based data and guiding inhibitor optimization. Herein, a HPLC-MS based protocol was developed to determine inhibitor cellular bioavailability. This generalizable protocol allows determination of the accurate intracellular concentrations and characterization of various properties of inhibitors including the extra- and intracellular stability, the dose- and time-dependence of the intracellular concentrations, the cell permeability, and the nonspecific binding with the cell culture plates, the extracellular matrices, and the cell membrane. The inhibitors of the protein-protein interactions, bromodomains, and the ß-catenin/B-cell lymphoma 9 (BCL9) interaction were used to examine the protocol, and the cellular bioavailability of the inhibitors in cancer cells was determined. High nonspecific binding and low cellular uptake were observed for two bromodomain inhibitors. The two ß-catenin/BCL9 inhibitors had low nonspecific binding but different cellular uptake. These inhibitors exhibited different stability kinetics in cells.

Structure-Based Design of 1,4-Dibenzoylpiperazines as ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction Inhibitors.

A small-molecule inhibitor with a 1,4-dibenzoylpiperazine scaffold was designed to match the critical binding elements in the ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction interface. Inhibitor optimization led to a potent inhibitor that can disrupt the ß-catenin/BCL9 interaction and exhibit 98-fold selectivity over the ß-catenin/cadherin interaction. The binding mode of new inhibitors was characterized by structure-activity relationships and site-directed mutagenesis studies. Cell-based studies demonstrated that this series of inhibitors can selectively suppress canonical Wnt signaling and inhibit growth of Wnt/ß-catenin-dependent cancer cells.

EGFR phosphorylates FAM129B to promote Ras activation.

Ras GTPase-activating proteins (GAPs) are important regulators for Ras activation, which is instrumental in tumor development. However, the mechanism underlying this regulation remains elusive. We demonstrate here that activated EGFR phosphorylates the Y593 residue of the protein known as family with sequence similarity 129, member B (FAM129B), which is overexpressed in many types of human cancer. FAM129B phosphorylation increased the interaction between FAM129B and Ras, resulting in reduced binding of p120-RasGAP to Ras. FAM129B phosphorylation promoted Ras activation, increasing ERK1/2- and PKM2-dependent ß-catenin transactivation and leading to the enhanced glycolytic gene expression and the Warburg effect; promoting tumor cell proliferation and invasion; and supporting brain tumorigenesis. Our studies unearthed a novel and important mechanism underlying EGFR-mediated Ras activation in tumor development.

Rational design of selective small-molecule inhibitors for ß-catenin/B-cell lymphoma 9 protein-protein interactions.

Selective inhibition of α-helix-mediated protein-protein interactions (PPIs) with small organic molecules provides great potential for the discovery of chemical probes and therapeutic agents. Protein Data Bank data mining using the HippDB database indicated that (1) the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix had few orientations when interacting with the second protein and (2) the hot spot pockets of PPI complexes had different sizes, shapes, and chemical groups when interacting with the same hydrophobic projecting hot spots of α-helix. On the basis of these observations, a small organic molecule, 4'-fluoro-N-phenyl-[1,1'-biphenyl]-3-carboxamide, was designed as a generic scaffold that itself directly mimics the binding mode of the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix. Convenient decoration of this generic scaffold led to the selective disruption of α-helix-mediated PPIs. A series of small-molecule inhibitors selective for ß-catenin/B-cell lymphoma 9 (BCL9) over ß-catenin/cadherin PPIs was designed and synthesized. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This new class of inhibitors can selectively disrupt ß-catenin/BCL9 over ß-catenin/cadherin PPIs, suppress the transactivation of canonical Wnt signaling, downregulate the expression of Wnt target genes, and inhibit the growth of Wnt/ß-catenin-dependent cancer cells.

Discovery of Selective Small-Molecule Inhibitors for the ß-Catenin/T-Cell Factor Protein-Protein Interaction through the Optimization of the Acyl Hydrazone Moiety.

Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the ß-catenin/Tcf protein-protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor optimization resulted in chemically stable derivatives that disrupt the ß-catenin/Tcf PPI. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This series of inhibitors with a new scaffold exhibits dual selectivity for ß-catenin/Tcf over ß-catenin/cadherin and ß-catenin/APC PPIs. One derivative of this series suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells. This compound represents a solid starting point for the development of potent and selective ß-catenin/Tcf inhibitors.

AlphaScreen selectivity assay for ß-catenin/B-cell lymphoma 9 inhibitors.

The aberrant formation of the ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein complex is the driving force for many diseases, including cancer. Crystallographic analyses demonstrate that the surface area in ß-catenin for interacting with BCL9 is overlapped with that for the ß-catenin/E-cadherin interaction. In this study, a robust AlphaScreen selectivity assay was developed to quantify inhibitor potency for the ß-catenin/BCL9 interaction and selectivity for ß-catenin/BCL9 over ß-catenin/E-cadherin interactions. A pilot screen was performed to demonstrat the feasibility of this assay. This selectivity assay is highly sensitive and suitable for adaptation to high-throughput screening. The establishment of this assay lays the foundation for the discovery of selective inhibitors specific for ß-catenin/BCL9 interactions.

The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases.

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity.

nNOS inhibition during profound asphyxia reduces seizure burden and improves survival of striatal phenotypic neurons in preterm fetal sheep.

Basal ganglia injury after hypoxia-ischemia remains common in preterm infants, and is closely associated with later cerebral palsy. In the present study we tested the hypothesis that a highly selective neuronal nitric oxide synthase (nNOS) inhibitor, JI-10, would improve survival of striatal phenotypic neurons after profound asphyxia, and that the subsequent seizure burden and recovery of EEG are associated with neural outcome. 24 chronically instrumented preterm fetal sheep were randomized to either JI-10 (3 ml of 0.022 mg/ml, n = 8) or saline (n = 8) infusion 15 min before 25 min complete umbilical cord occlusion, or saline plus sham-occlusion (n = 8). Umbilical cord occlusion was associated with reduced numbers of calbindin-28k-, GAD-, NPY-, PV-, Calretinin- and nNOS-positive striatal neurons (p < 0.05 vs. sham occlusion) but not ChAT-positive neurons. JI-10 was associated with increased numbers of calbindin-28k-, GAD-, nNOS-, NPY-, PV-, Calretinin- and ChAT-positive striatal neurons (p < 0.05 vs. saline + occlusion). Seizure burden was strongly associated with loss of calbindin-positive cells (p < 0.05), greater seizure amplitude was associated with loss of GAD-positive cells (p < 0.05), and with more activated microglia in the white matter tracts (p < 0.05). There was no relationship between EEG power after 7 days recovery and total striatal cell loss, but better survival of NPY-positive neurons was associated with lower EEG power. In summary, these findings suggest that selective nNOS inhibition during asphyxia is associated with protection of phenotypic striatal projection neurons and has potential to help reduce basal ganglia injury in some premature babies.