OPTN/SRTR 2022 Annual Data Report: Kidney.

The year 2022 had continued successes and challenges for the field of kidney transplantation, as the community adapted to ongoing surges of the COVID-19 pandemic and broader geographic organ distribution. The total number of kidney transplants in the United States reached a record count of 26,309, driven by continued growth in deceased donor kidney transplants (DDKTs). The total number of candidates listed for DDKT rose slightly in 2022 but remained below 2019 listing levels, with 12.4% of candidates having been waiting 5 years or longer. Following the height of the COVID-19 pandemic, pretransplant mortality in 2022 declined across age, race and ethnicity, sex, and blood type groups. Pretransplant mortality continued to vary substantially by donation service area. The proportion of deceased donor kidneys recovered but not used for transplant (nonuse rate) rose to a high of 26.7% overall, with greater nonuse of biopsied kidneys (39.8%), kidneys from donors aged 55 years or older (54.7%), and kidneys with a kidney donor profile index (KDPI) of 85% or greater (71.3%). Nonuse of kidneys from donors who are hepatitis C virus (HCV) antibody positive rose to 30.2% but only slightly exceeded that of HCV antibody-negative donors. Disparities in access to living donor kidney transplant (LDKT) persist, especially for non-White and publicly insured patients. Delayed graft function continues an upward trend and occurred in 26.3% of adult kidney transplants in 2022. Five-year graft survival after LDKT compared with DDKT was 90.0% versus 81.4% for recipients aged 18-34 years and 80.8% versus 67.8% for recipients aged 65 years or older, respectively. The total number of pediatric kidney transplants performed in 2022 decreased to 705, its lowest point in the past decade; 502 (71.2%) were DDKTs and 203 (28.8%) were LDKTs. Among pediatric recipients, LDKT remains low, with continued racial disparities. The rate of DDKT among pediatric candidates has decreased by almost 25% since 2011. Congenital anomalies of the kidney and urinary tract remain the leading primary kidney disease diagnosis among pediatric candidates with a reported diagnosis. Most pediatric deceased donor recipients received a kidney from a donor with a KDPI of less than 35%. The rate of delayed graft function was 5.8% in 2022 and has been stable over the past decade. Long-term graft survival continues to improve, with superior outcomes for living donor transplant recipients.

OPTN/SRTR 2021 Annual Data Report: Kidney.

The year 2021 marked both successes and challenges for the field of kidney transplantation, in the context of the ongoing COVID-19 pandemic and broader geographic organ distribution. The total number of kidney transplants in the United States reached a record count of 25,487, driven by growth in deceased donor kidney transplants. The total number of candidates listed for deceased donor kidney transplant rose slightly in 2021 but remained below 2019 listing levels, with nearly 10% of candidates having been waiting 5 years or longer. Pretransplant mortality declined slightly among candidates of Black, Hispanic, and other races, in parallel with increasing numbers of Black and Hispanic transplant recipients. In the context of broader organ sharing, there is growing disparity in pretransplant mortality among non-metropolitan compared with metropolitan residents. The proportion of deceased donor kidneys recovered but not used for transplant (nonuse rate) rose to a high of 24.6% overall, with greater nonuse among biopsied kidneys (35.9%), kidneys from donors aged 55 years or older (51.1%), and kidneys with kidney donor profile index (KDPI) of 85% or greater (66.6%). Nonuse of kidneys from donors who are hepatitis C virus (HCV) antibody positive only slightly exceeded that of HCV antibody-negative donors. Disparities in access to living donor kidney transplant persists, especially for non-White and publicly insured patients. Delayed graft function continues an upward trend and occurred in 24% of adult kidney transplants in 2021. Five-year graft survival after living compared with deceased donor transplant was 88.6% versus 80.7% for recipients aged 18-34 years, and 82.1% versus 68.0% for recipients aged 65 years or older. The total number of pediatric kidney transplants performed increased to 820 in 2021, the highest number since 2010. Despite numerous efforts, living donor kidney transplant remains low among pediatric recipients, with continued racial disparities. The rate of deceased donor transplants among pediatric candidates recovered in 2021 from a low in 2020. Congenital anomalies of the kidney and urinary tract remain the leading primary kidney disease diagnosis among pediatric candidates. Most pediatric deceased donor recipients receive a kidney from a donor with KDPI less than 35%. Graft survival continues to improve, with superior outcomes for living donor transplant recipients.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes - Part 2.

This Letter details our efforts to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core which lead to the discovery of two novel tricyclic cores: a 7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine core and 2,4-dimethylthieno[2,3-b:5,4-c']dipyridine core. Both tricyclic cores displayed low nanomolar potency against the human M4 receptor.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes.

This Letter details our efforts to develop new M4 PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M4.

Discovery of a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide M4 positive allosteric modulator (PAM) chemotype.

This Letter details our efforts to discover structurally unique M4 PAMs containing 5,6-heteroaryl ring systems. In an attempt to improve the DMPK profiles of the 2,3-dimethyl-2H-indazole-5-carboxamide and 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide cores, we investigated a plethora of core replacements. This exercise identified a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide core that provided improved M4 PAM activity and CNS penetration.

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M4 PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M4 PAM activity and improved clearance and protein binding profiles.

M1 Muscarinic Receptors Modulate Fear-Related Inputs to the Prefrontal Cortex: Implications for Novel Treatments of Posttraumatic Stress Disorder.

BACKGROUND: The prefrontal cortex (PFC) integrates information from multiple inputs to exert top-down control allowing for appropriate responses in a given context. In psychiatric disorders such as posttraumatic stress disorder, PFC hyperactivity is associated with inappropriate fear in safe situations. We previously reported a form of muscarinic acetylcholine receptor (mAChR)-dependent long-term depression in the PFC that we hypothesize is involved in appropriate fear responding and could serve to reduce cortical hyperactivity following stress. However, it is unknown whether this long-term depression occurs at fear-related inputs. METHODS: Using optogenetics with extracellular and whole-cell electrophysiology, we assessed the effect of mAChR activation on the synaptic strength of specific PFC inputs. We used selective pharmacological tools to assess the involvement of M1 mAChRs in conditioned fear extinction in control mice and in the stress-enhanced fear-learning model. RESULTS: M1 mAChR activation induced long-term depression at inputs from the ventral hippocampus and basolateral amygdala but not from the mediodorsal nucleus of the thalamus. We found that systemic M1 mAChR antagonism impaired contextual fear extinction. Treatment with an M1 positive allosteric modulator enhanced contextual fear extinction consolidation in stress-enhanced fear learning-conditioned mice. CONCLUSIONS: M1 mAChRs dynamically modulate synaptic transmission at two PFC inputs whose activity is necessary for fear extinction, and M1 mAChR function is required for proper contextual fear extinction. Furthermore, an M1 positive allosteric modulator enhanced the consolidation of fear extinction in the stress-enhanced fear-learning model, suggesting that M1 positive allosteric modulators may provide a novel treatment strategy to facilitate exposure therapy in the clinic for the treatment of posttraumatic stress disorder.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Discoidin domain receptor 1 kinase activity is required for regulating collagen IV synthesis.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds to and is activated by collagens. DDR1 expression increases following kidney injury and accumulating evidence suggests that it contributes to the progression of injury. To this end, deletion of DDR1 is beneficial in ameliorating kidney injury induced by angiotensin infusion, unilateral ureteral obstruction, or nephrotoxic nephritis. Most of the beneficial effects observed in the DDR1-null mice are attributed to reduced inflammatory cell infiltration to the site of injury, suggesting that DDR1 plays a pro-inflammatory effect. The goal of this study was to determine whether, in addition to its pro-inflammatory effect, DDR1 plays a deleterious effect in kidney injury by directly regulating extracellular matrix production. We show that DDR1-null mice have reduced deposition of glomerular collagens I and IV as well as decreased proteinuria following the partial renal ablation model of kidney injury. Using mesangial cells isolated from DDR1-null mice, we show that these cells produce significantly less collagen compared to DDR1-null cells reconstituted with wild type DDR1. Moreover, mutagenesis analysis revealed that mutations in the collagen binding site or in the kinase domain significantly reduce DDR1-mediated collagen production. Finally, we provide evidence that blocking DDR1 kinase activity with an ATP-competitive small molecule inhibitor reduces collagen production. In conclusion, our studies indicate that the kinase activity of DDR1 plays a key role in DDR1-induced collagen synthesis and suggest that blocking collagen-mediated DDR1 activation may be beneficial in fibrotic diseases.

Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets.

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.

Synthesis of Non-natural, Frame-Shifted Isoprenoid Diphosphate Analogues.

A set of synthetic approaches was developed and applied to the synthesis of eight frame-shifted isoprenoid diphosphate analogues. These analogues were designed to increase or decrease the methylene units between the double bonds and/or the pyrophosphate moieties of the isoprenoid structure. Evaluation of mammalian GGTase-I and FTase revealed that small structural changes can result in substantial changes in substrate activity.

Identification of the minimum PAR4 inhibitor pharmacophore and optimization of a series of 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazoles.

This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490-525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by ∼50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate.

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)-1H-indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ-Thrombin.

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based 3. Of these, 9j (PAR4 IC50 = 445 nM, PAR1 response IC50 > 30 µM) and 10h (PAR4 IC50 = 179 nM, PAR1 response IC50 > 30 µM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.

Exploration of GGTase-I substrate requirements. Part 1: Synthesis and biochemical evaluation of novel aryl-modified geranylgeranyl diphosphate analogs.

Protein geranylgeranylation is a type of post-translational modification that aids in the localization of proteins to the plasma member where they elicit cellular signals. To better understand the isoprenoid requirements of GGTase-I, a series of aryl-modified geranylgeranyl diphosphate analogs were synthesized and screened against mammalian GGTase-I. Of our seven-member library of compounds, six analogs proved to be substrates of GGTase-I, with 6d having a krel=1.93 when compared to GGPP (krel=1.0).

Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

Protein prenylation is a type of post-translational modification that aids certain proteins in localizing to the plasma member where they activate cell signaling. To better understand the isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of GGTase-I, with 11d having a krel=0.95 when compared to GGPP (krel=1.0).

Synthesis and characterization of a series of chiral alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a series of chiral alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel potent and selective dopamine D4 receptor antagonist with selectivity against the other dopamine receptors tested (<10% inhibition at 1µM against D1, D2L, D2S, D3, and D5).

Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a novel series of (R)-4,4-difluoropiperidine core scaffold as dopamine receptor 4 (D4) antagonists. A series of compounds from this scaffold are highly potent against the D4 receptor and selective against the other dopamine receptors. In addition, we were able to confirm the active isomer as the (R)-enantiomer via an X-ray crystal structure.