Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

Mechanisms of Selective Autophagy.

Selective autophagy is the lysosomal degradation of specific intracellular components sequestered into autophagosomes, late endosomes, or lysosomes through the activity of selective autophagy receptors (SARs). SARs interact with autophagy-related (ATG)8 family proteins via sequence motifs called LC3-interacting region (LIR) motifs in vertebrates and Atg8-interacting motifs (AIMs) in yeast and plants. SARs can be divided into two broad groups: soluble or membrane bound. Cargo or substrate selection may be independent or dependent of ubiquitin labeling of the cargo. In this review, we discuss mechanisms of mammalian selective autophagy with a focus on the unifying principles employed in substrate recognition, interaction with the forming autophagosome via LIR-ATG8 interactions, and the recruitment of core autophagy components for efficient autophagosome formation on the substrate.

A Diversity of Selective Autophagy Receptors Determines the Specificity of the Autophagy Pathway.

The clearance of surplus, broken, or dangerous components is key for maintaining cellular homeostasis. The failure to remove protein aggregates, damaged organelles, or intracellular pathogens leads to diseases, including neurodegeneration, cancer, and infectious diseases. Autophagy is the evolutionarily conserved pathway that sequesters cytoplasmic components in specialized vesicles, autophagosomes, which transport the cargo to the degradative compartments (vacuoles or lysosomes). Research during the past few decades has elucidated how autophagosomes engulf their substrates selectively. This type of autophagy involves a growing number of selective autophagy receptors (SARs) (e.g., Atg19 in yeasts, p62/SQSTM1 in mammals), which bind to the cargo and simultaneously engage components of the core autophagic machinery via direct interaction with the ubiquitin-like proteins (UBLs) of the Atg8/LC3/GABARAP family and adaptors, Atg11 (in yeasts) or FIP200 (in mammals). In this Review, we critically discuss the biology of the SARs with special emphasis on their interactions with UBLs.

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Immunity priming uncouples the growth-defense trade-off in tomato.

Plants have developed an array of mechanisms to protect themselves against pathogen invasion. The deployment of defense mechanisms is imperative for plant survival, but can come at the expense of plant growth, leading to the 'growth-defense trade-off' phenomenon. Following pathogen exposure, plants can develop resistance to further attack. This is known as induced resistance, or priming. Here, we investigated the growth-defense trade-off, examining how defense priming via systemic acquired resistance (SAR), or induced systemic resistance (ISR), affects tomato development and growth. We found that defense priming can promote, rather than inhibit, plant development, and that defense priming and growth trade-offs can be uncoupled. Cytokinin response was activated during induced resistance, and found to be required for the observed growth and disease resistance resulting from ISR activation. ISR was found to have a stronger effect than SAR on plant development. Our results suggest that growth promotion and induced resistance can be co-dependent, and that, in certain cases, defense priming can drive developmental processes and promote plant yield.

Identification of vital chemical information via visualization of graph neural networks.

Qualitative or quantitative prediction models of structure-activity relationships based on graph neural networks (GNNs) are prevalent in drug discovery applications and commonly have excellently predictive power. However, the network information flows of GNNs are highly complex and accompanied by poor interpretability. Unfortunately, there are relatively less studies on GNN attributions, and their developments in drug research are still at the early stages. In this work, we adopted several advanced attribution techniques for different GNN frameworks and applied them to explain multiple drug molecule property prediction tasks, enabling the identification and visualization of vital chemical information in the networks. Additionally, we evaluated them quantitatively with attribution metrics such as accuracy, sparsity, fidelity and infidelity, stability and sensitivity; discussed their applicability and limitations; and provided an open-source benchmark platform for researchers. The results showed that all attribution techniques were effective, while those directly related to the predicted labels, such as integrated gradient, preferred to have better attribution performance. These attribution techniques we have implemented could be directly used for the vast majority of chemical GNN interpretation tasks.

Identifying components of the Shewanella phage LambdaSo lysis system.

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.

Nature should be the model for microbial sciences.

Until recently, microbiologists have relied on cultures to understand the microbial world. As a result, model organisms have been the focus of research into understanding Bacteria and Archaea at a molecular level. Diversity surveys and metagenomic sequencing have revealed that these model species are often present in low abundance in the environment; instead, there are microbial taxa that are cosmopolitan in nature. Due to the numerical dominance of these microorganisms and the size of their habitats, these lineages comprise mind-boggling population sizes upward of 1028 cells on the planet. Many of these dominant groups have cultured representatives and have been shown to be involved in mediating key processes in nature. Given their importance and the increasing need to understand changes due to climate change, we propose that members of Nitrosophaerota (Nitrosopumilus maritimus), SAR11 (Pelagibacter ubique), Hadesarchaeia, Bathyarchaeia, and others become models in the future. Abundance should not be the only measure of a good model system; there are other organisms that are well suited to advance our understanding of ecology and evolution. For example, the most well-studied symbiotic bacteria, like Buchnera, Aliivibrio, and Rhizobium, should be models for understanding host-associations. Also, there are organisms that hold new insights into major transitions in the evolution of life on the planet like the Asgard Archaea (Heimdallarchaeia). Innovations in a variety of in situ techniques have enabled us to circumvent culturing when studying everything from genetics to physiology. Our deepest understanding of microbiology and its impact on the planet will come from studying these microbes in nature. Laboratory-based studies must be grounded in nature, not the other way around.

1,2,4,5-Tetraoxane derivatives/hybrids as potent antimalarial endoperoxides: Chronological advancements, structure-activity relationship (SAR) studies and future perspectives.

Malaria is a life-threatening disease that affects tropical and subtropical regions worldwide. Various drugs were used to treat malaria, including artemisinin and derivatives, antibiotics (tetracycline, doxycycline), quinolines (chloroquine, amodiaquine), and folate antagonists (sulfadoxine and pyrimethamine). Since the malarial parasites developed drug resistance, there is a need to develop new chemical entities with high efficacy and low toxicity. In this context, 1,2,4,5-tetraoxanes emerged as an essential scaffold and have shown promising antimalarial activity. To improve activity and overcome resistance to various antimalarial drugs; 1,2,4,5-tetraoxanes were fused with various aryl/heteroaryl/alicyclic/spiro moieties (steroid-based 1,2,4,5-tetraoxanes, triazine-based 1,2,4,5-tetraoxanes, aminoquinoline-based 1,2,4,5-tetraoxanes, dispiro-based 1,2,4,5-tetraoxanes, piperidine-based 1,2,4,5-tetraoxanes and diaryl-based 1,2,4,5-tetraoxanes). The present review aims to focus on covering the relevant literature published during the past 30 years (1992-2022). We summarize the most significant in vitro, in vivo results and structure-activity relationship studies of 1,2,4,5-tetraoxane-based hybrids as antimalarial agents. The structural evolution of different hybrids can provide the framework for the future development of 1,2,4,5-tetraoxane-based hybrids to treat malaria.

Local and whole-body SAR in UHF body imaging: Implications for SAR matrix compression.

PURPOSE: Transmit arrays for body imaging have characteristics of both volume and local transmit coils. This study evaluates two specific absorption rate (SAR) aspects, local and whole-body SAR, of arrays for body imaging at 7 T and also for a 3 T birdcage. METHODS: Simulations were performed for six antenna arrays at 7 T and one 3 T birdcage. Local SAR matrices and the whole-body SAR matrix were computed and evaluated with random shims. A set of reduced local SAR matrices was determined by removing all matrices dominated by the whole-body SAR matrix. RESULTS: The results indicate that all RF transmit coils for body imaging in this study are constrained by the local SAR limit. The ratio between local and whole-body SAR is nevertheless smaller for arrays with large FOV, as these arrays also expose a larger part of the human body. By using the whole-body SAR matrix, the number of local SAR matrices can be reduced (e.g., 33.3% matrices remained for an 8-channel local array and 89.7% for a 30-channel remote array; 12.1% for the 3 T birdcage). CONCLUSION: For transmit antenna arrays used for body imaging at 7 T as well as for the 3 T birdcage, all evaluated cases show that the local SAR limit was reached before reaching the whole-body SAR limit. Nevertheless, the whole-body SAR matrix can be used to reduce the number of local SAR matrices, which is important to reduce memory and computing time for a virtual observation points (VOP) compression. This step can be included as a pre-compression prior to a VOP compression.

Multiphoton simultaneous multislice imaging.

PURPOSE: To develop multiphoton excitation techniques for simultaneous multislice (SMS) imaging and evaluate their performance and specific absorption rate (SAR) benefit. To improve multiphoton SMS reconstruction quality with a novel CAIPIRINHA (controlled aliasing in parallel imaging results in higher acceleration) design. THEORY AND METHODS: When a conventional single-slice RF field is applied together with an oscillating gradient field, the two can combine to generate multiphoton excitation at multiple discrete spatial locations. Because the conventional RF is reused at multiple spatial locations, multiphoton excitation offers reduced SAR for SMS applications. CAIPIRINHA shifts are often used to improve parallel-imaging acceleration. Interestingly, CAIPIRINHA-type shifts can be obtained for multiphoton SMS by updating the oscillating gradient phase at every phase encode. In this work, both a gradient-echo and a spin-echo sequence with multiphoton CAIPIRINHA-SMS excitation pulses are implemented for in vivo human imaging at 3 T. RESULTS: For three slices, multiphoton SMS provides a 51% reduction in SAR compared with conventional superposition SMS, whereas for five slices, SAR is reduced by 66%. Multiphoton SMS outperforms PINS (power independent of number of slices) and MultiPINS in terms of SAR reduction especially when the pulse duration is short, slices are thin, and/or the slice spacing is large. A custom CAIPIRINHA phase-encoding design for multiphoton SMS significantly improves reconstruction quality. CONCLUSION: Multiphoton SMS excitation can be obtained by combining conventional single-slice RF pulses with an oscillating gradient and offers significant SAR benefits compared with conventional superposition SMS. A novel CAIPIRINHA design allows higher multiband factors for multiphoton SMS imaging.

The restricted SAR protocol: A method to assess MRI coil prototypes in an unconditionally safe manner.

PURPOSE: Testing an RF coil prototype on subjects involves laborious verifications to ensure its safety. In particular, it requires preliminary electromagnetic simulations and their validations on phantoms to accurately predict the specific absorption rate (SAR). For coil design validation with a simpler safety procedure, the restricted SAR (rS) mode is proposed, enabling representative first experiments in vivo. The goal of the developed approach is to accelerate the transition of a custom coil system from prototype to clinical use. METHODS: The restricted specific absorption rate (SAR) (rS) mode imposes a radical limitation on the transmitted RF power based on a worst-case scenario of local RF power absorption. The limitations used are independent of the SAR spatial distribution, making this approach unconditionally safe. The developed rS protocol contains the sequences required for coil evaluation and satisfies the imposed rS conditions. It provides a quantitative characterization of the coil transmission and reception profiles and a qualitative evaluation of the anatomical images. Protocol validation was performed on commercial and pre-industrial prototype coils on a small cohort of healthy volunteers. RESULTS: The proposed rS protocol enables coil evaluation within an acquisition time compatible with common clinical protocol duration. The total time of all evaluation steps does not exceed 17 min. At the same time, the global SAR remains 100 times less than the International Electrotechnical Commission safety limit for played sequences. CONCLUSION: The rS protocol allows characterizing and comparing coil prototypes on volunteers without extensive electromagnetic calculations and phantom validations in an unconditionally safe way.

Comparison of tight-fitting 7T parallel-transmit head array designs using excitation uniformity and local specific absorption rate metrics.

PURPOSE: We model the performance of parallel transmission (pTx) arrays with 8, 16, 24, and 32 channels and varying loop sizes built on a close-fitting helmet for brain imaging at 7 T and compare their local specific absorption rate (SAR) and flip-angle performances to that of birdcage coil (used as a baseline) and cylindrical 8-channel and 16-channel pTx coils (single-row and dual-row). METHODS: We use the co-simulation approach along with MATLAB scripting for batch-mode simulation of the coils. For each coil, we extracted B1 + maps and SAR matrices, which we compressed using the virtual observation points algorithm, and designed slice-selective RF shimming pTx pulses with multiple local SAR and peak power constraints to generate L-curves in the transverse, coronal, and sagittal orientations. RESULTS: Helmet designs outperformed cylindrical pTx arrays at a constant number of channels in the flip-angle uniformity at a constant local SAR metric: up to 29% for 8-channel arrays, and up to 34% for 16-channel arrays, depending on the slice orientation. For all helmet arrays, increasing the loop diameter led to better local SAR versus flip-angle uniformity tradeoffs, although this effect was more pronounced for the 8-channel and 16-channel systems than the 24-channel and 32-channel systems, as the former have more limited degrees of freedom and therefore benefit more from loop-size optimization. CONCLUSION: Helmet pTx arrays significantly outperformed cylindrical arrays with the same number of channels in local SAR and flip-angle uniformity metrics. This improvement was especially pronounced for non-transverse slice excitations. Loop diameter optimization for helmets appears to favor large loops, compatible with nearest-neighbor decoupling by overlap.

Local SAR management strategies to use two-channel RF shimming for fetal MRI at 3 T.

PURPOSE: This study evaluates the imaging performance of two-channel RF-shimming for fetal MRI at 3 T using four different local specific absorption rate (SAR) management strategies. METHODS: Due to the ambiguity of safe local SAR levels for fetal MRI, local SAR limits for RF shimming were determined based on either each individual's own SAR levels in standard imaging mode (CP mode) or the maximum SAR level observed across seven pregnant body models in CP mode. Local SAR was constrained either indirectly by further constraining the whole-body SAR (wbSAR) or directly by using subject-specific local SAR models. Each strategy was evaluated by the improvement of the transmit field efficiency (average |B1 + |) and nonuniformity (|B1 + | variation) inside the fetus compared with CP mode for the same wbSAR. RESULTS: Constraining wbSAR when using RF shimming decreases B1 + efficiency inside the fetus compared with CP mode (by 12%-30% on average), making it inefficient for SAR management. Using subject-specific models with SAR limits based on each individual's own CP mode SAR value, B1 + efficiency and nonuniformity are improved on average by 6% and 13% across seven pregnant models. In contrast, using SAR limits based on maximum CP mode SAR values across seven models, B1 + efficiency and nonuniformity are improved by 13% and 25%, compared with the best achievable improvement without SAR constraints: 15% and 26%. CONCLUSION: Two-channel RF-shimming can safely and significantly improve the transmit field inside the fetus when subject-specific models are used with local SAR limits based on maximum CP mode SAR levels in the pregnant population.

Evaluation of coaxial dipole antennas as transceiver elements of human head array for ultra-high field MRI at 9.4T.

PURPOSE: The aim of this work is to evaluate a new eight-channel transceiver (TxRx) coaxial dipole array for imaging of the human head at 9.4T developed to improve specific absorption rate (SAR) performance, and provide for a more compact and robust alternative to the state-of-the art dipole arrays. METHODS: First, the geometry of a single coaxial element was optimized to minimize peak SAR and sensitivity to the load variation. Next, a multi-tissue voxel model was used to numerically simulate a TxRx array coil that consisted of eight coaxial dipoles with the optimal configuration. Finally, we compared the developed array to other human head dipole arrays. Results of numerical simulations were verified on a bench and in the scanner including in vivo measurements on a healthy volunteer. RESULTS: The developed eight-element coaxial dipole TxRx array coil showed up to 1.1times higher SAR-efficiency than a similar in geometry folded-end and fractionated dipole array while maintaining whole brain coverage and low sensitivity of the resonance frequency to variation in the head size. CONCLUSION: As a proof of concept, we developed and constructed a prototype of a 9.4T (400 MHz) human head array consisting of eight TxRx coaxial dipoles. The developed array improved SAR-efficiency and provided for a more compact and robust alternative to the folded-end dipole design. To the best of our knowledge, this is the first example of using coaxial dipoles for human head MRI at ultra-high field.

Hybrid algorithms for SAR matrix compression and the impact of post-processing on SAR calculation complexity.

PURPOSE: This study proposes faster virtual observation point (VOP) compression as well as post-processing algorithms for specific absorption rate (SAR) matrix compression. Furthermore, it shows the relation between the number of channels and the computational burden for VOP-based SAR calculation. METHODS: The proposed new algorithms combine the respective benefits of two different criteria for determining upper boundedness of SAR matrices by the VOPs. Comparisons of the old and new algorithms are performed for head coil arrays with various channel counts. The new post-processing algorithm is used to post-process the VOP sets of nine arrays, and the number of VOPs for a fixed median relative overestimation is compared. RESULTS: The new algorithms are faster than the old algorithms by a factor of two to more than 10. The compression efficiency (number of VOPs relative to initial number of SAR matrices) is identical. For a fixed median relative overestimation, the number of VOPs increases logarithmically with the number of RF coil channels when post-processing is applied. CONCLUSION: The new algorithms are much faster than previous algorithms. Post-processing is very beneficial for online SAR supervision of MRI systems with high channel counts, since for a given number of VOPs the relative SAR overestimation can be lowered.

Prognostic and Predictive Significance of Ki67 in Primary Non-metastatic or Recurrent Acral Melanoma: Evidence from a Multicenter Retrospective Study.

BACKGROUND: The purpose of this work was to investigate the prognostic significance of Ki67 in acral melanoma (AM). PATIENTS AND METHODS: Ki67 values in primary lesions (pKi67) of 481 patients with primary non-metastatic AM (primary cohort) from three tertiary hospitals and in recurrent lesions (rKi67) of 97 patients (recurrent cohort) were recorded. The associations of p/rKi67 with clinicopathological features and prognosis were analyzed. RESULTS: In the primary cohort, high pKi67 group tended to have more ulceration, pT4, lymph node metastasis (LNM), nodal macrometastases, and recurrence (all P < 0.05). Logistic regression analysis revealed that pKi67 was significantly associated with pT4 and LNM (P = 0.004 and 0.027, respectively). Furthermore, both 5-year overall survival (OS) and recurrence-free survival (RFS) rates in high pKi67 group were significantly worse than those in moderate and low pKi67 groups (OS 47.8% versus 55.7 versus 76.8%, P = 0.002; RFS: 27.1 versus 42.8 versus 61.8%, P < 0.001). Similarly, in the recurrent cohort, the 5-year survival after recurrence (SAR) rates in high rKi67 group was significantly worse than those in moderate and low rKi67 groups (31.7 versus 47.4 versus 75%; P = 0.026). Stratified analysis also indicated a significant survival difference among pKi67 groups within various subgroups. Most importantly, multivariate Cox analysis demonstrated that pKi67 could be independently associated with OS and RFS, as well as rKi67 for SAR (all P < 0.05). CONCLUSIONS: A high Ki67 value was significantly associated with adverse pathological and prognostic features in both primary and recurrent AM cohorts. Ki67 should be routinely evaluated to guide risk stratification and prognostic prediction.

An efficient method for detecting membrane protein oligomerization and complex using 05SAR-PAGE.

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions.

Chemical Predictive Modelling and Natural Product-based Divergent Synthesis - Design of Type B PPAPs with Nanomolar Activities against MRSA.

In response to the pressing global challenge of antibiotic resistance, time efficient design and synthesis of novel antibiotics are of immense need. Polycyclic polyprenylated acylphloroglucinols (PPAP) were previously reported to effectively combat a range of gram-positive bacteria. Although the exact mode of action is still not clear, we conceptualized a late-stage divergent synthesis approach to expand our natural product-based PPAP library by 30 additional entities to perform SAR studies against methicillin-resistant Staphylococcus aureus (MRSA). Although at this point only data from cellular assays are available and understanding of molecular drug-target interactions are lacking, the experimental data were used to generate 3D-QSAR models via an artificial intelligence training and to identify a common pharmacophore model. The experimentally validated QSAR model enabled the estimation of anti-MRSA activities of a virtual compound library consisting of more than 100,000 in-silico generated B PPAPs, out of which the 20 most promising candidates were synthesized. These novel PPAPs revealed significantly improved cellular activities against MRSA with growth inhibition down to concentrations less than 1 µm.

Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine.

Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.